CA2420459A1

CA2420459A1 - Novel dermatophagoides nucleic acid molecules, proteins and uses thereof

Info

Publication number: CA2420459A1
Application number: CA002420459A
Authority: CA
Inventors: Catherine A. Mccall; Shirley Wu Hunter; Eric R. Weber
Original assignee: Individual
Current assignee: Heska Corp
Priority date: 2000-09-14
Filing date: 2001-09-14
Publication date: 2002-03-21
Also published as: AU2001292661A1; WO2002022807A3; EP1328625A2; JP2004529605A; WO2002022807A2

Abstract

The present invention relates to high molecular weight Dermatophagoides proteins, nucleic acid molecules encoding such proteins, and therapeutic and diagnostic reagents derived from such proteins.

Description

NOVEL DERMATOPHAGOIDES NUCLEIC ACID MOLECULES, PROTEINS AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to high molecular weight DeYfnatophagoides proteins, nucleic acid molecules and therapeutic and diagnostic reagents derived from such proteins.
BACKGROUND OF THE INVENTION
Immunoglobulin E (IgE) mediated allergic symptoms afflict many animals.
IgE antibody production in an animal can induce pathogenic IgE responses including, for example, atopic disease, asthma and rhinitis. Allergens are proteins or peptides characterized by their ability to induce a pathogenic IgE response in susceptible individuals.
House dust mite (e.g., Derrnatophagoides fariu.ae and Dernzatophagoides pteYOrzyssircus; l7er f and Der p, respectively) allergens are major causative agents associated With IgE-mediated pathogenesis. Previous investigators have identified two major groups of dust mite allergens in humans, group I (Der f I and Der p I, Mr 25,000) and group 2 (Der f II and Der p II, Mr 14,000); reviewed in Chapman, et al., Allergy, vol. 52, pp.37-379, 1997. Prior investigators have disclosed nucleotide and/or annino acid sequences for: Der f I, Der f II, Der p I and Der p II, U.S.
Patent No.
5,552,142, to Thomas et al., issued September 3, 1996, U.S. Patent No.
5,460,977, to Ando et al., issued October 24, 1995, PCT Patent Publication No. WO 95/28424, by Chen et al., published October 26, 1995, U.S. Patent No. 5,433,948, to Thomas et al., issued July 18, 1995, PCT Patent Publication No. WO 93/08279, by Garmen et al., published March 4, 1993, or Chapman, ibid.; Der p III, PCT Patent Publication No.
WO 95/15976, by Thomas et al., published June 15, 1995; Der p VII, PCT Patent Publication No. WO 94/20614, by Thomas et al., published September 15, 1994; a kilodalton (kd) Der f allergen, U.S. Patent No. 5,405,758, to Oka et al., issued April 11, 1995, U.S. Patent No. 5,314,991, to Oka et al., issued May 24, 1994; a 70-kd Des- f allergen which is a heat shock protein (Hsp70), Aki et al., J. Biochef7z., vol. 115, pp.
435-440, 1994; or Noli et al., Vet. Imfzzunol. lyvmmaopath., vol. 52, pp. 147-157, 1996;
and a 98-kd Der f paramyosin-like allergen, Tsai et al, J. Allergy Clirz.
Imrnuzzol., vol.

102, pp. 295-303, 1998. None of these published sequences indicates, suggests or predicts any of the mite allergic nucleic acid molecules or proteins of the present invention, nor the relevance of such proteins as being immunoreactive with IgE
antibodies in canine, feline, or human sera.
Products and processes of the present invention are needed in the art that provide specific detection and treatment of mite allergy. .
SUMMARY OF THE INVENTION
The present invention relates to novel proteins having molecular weights of about 60 kilodaltons (kd or kD), 70 kD, or from about 98 kD to about 109 kD.
Such proteins include at least one epitope of a protein allergen of a mite of the genus Dermatophagoides and are designated herein as Def° HMW-map proteins.
Preferred proteins are Dermatophagoides farinae or Derrnatoplaagoides ptet~onyssius proteins.
The present invention also provides proteins that are fragments or peptides of full-length or mature proteins, as well as antibodies, rnimetopes or muteins of any of such proteins. The present invention also provides nucleic acid molecules encoding any of such proteins, as well as complements thereof. The present invention also includes methods to obtain such proteins, nucleic acid molecules, antibodies, mimetopes or muteins, as well as methods to use such compounds in diagnostic or therapeutic applications. 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 1X 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
N0:14, SEQ m N0:16, SEQ ID N0:17, SEQ m N0:19, SEQ ID N0:20, SEQ ID

N0:22, SEQ m N0:34, SEQ m N0:36, SEQ m N0:37, SEQ ID N0:39, SEQ m N0:40, SEQ m N0:42, SEQ ID N0:43, SEQ ID N0:45, and a nucleic acid sequence encoding a protein comprising the amino acid sequence of SEQ ID N0:33 and a complement thereof; 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 recombinant molecules, recombinant viruses and recombinant cells comprising such nucleic acid sequences as well as methods to produce them.
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 1X 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 m N0:16, SEQ ID N0:19, SEQ ID N0:22, SEQ ID
N0:36, SEQ ID N0:39, SEQ ID N0:42, SEQ ID N0:45, and a complement of a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID
N0:33; 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.
An isolated protein of the present invention can also be encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions with the complement of a nucleic acid molecule that encodes a protein having at least one of the following amino acid sequences: SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ m N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID
NO:10, SEQ m NO:l 1, SEQ ID N0:12, SEQ ID N0:13, SEQ ID NO:15, SEQ ID
N0:18, SEQ m N0:21, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:29, SEQ m N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:35, SEQ ID
N0:38, SEQ ID N0:41, and SEQ m N0:44. 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. Such 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. Also included 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 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. Such 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. Such 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 ~3-elimination of peptides; (b) the epitope is resistant to Proteinase-I~
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. Also included is an isolated antibody that selectively binds such a non-proteinaceous epitope as well as derivatives of such an 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.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 illustrates high molecular weight Der f proteins resolved by 12% Tris-Glycine SDS-PAGE.

Fig. 2 illustrates an about 60 kD Def~ f protein resolved by 14% Tris-Glycine SDS-PAGE.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides for isolated proteins having molecular weights ranging from about 60 kilodaltons (kD) to about 109 kD, that include at least one epitope of a protein allergen of a mite of the genus Dermatophagoides, in particular a mite of the species Dermatophagoides farinae and/or Derma.tophagoides pteYOnyssius.
Such proteins are referred to herein as Der HMW-map proteins. The present invention further includes methods to isolate and identify nucleic acid molecules encoding DeYHMW-map proteins, antibodies directed against Der HMW-map proteins and inhibitors of Der HMW-map protein activity. As used herein, the term isolated Der HMW-map proteins refers to Der HMW-map proteins derived from Dermatophagoides, and more preferably from Denrnatophagoides far°iT2ae and/or Dermatophagoides pteronyssius and, as such, can be obtained from its natural source or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. Also included in the present invention is the use of this protein and antibodies in a method to detect immunoglobulin that specifically binds to DeY
HMW-map proteins, to treat pathogenesis against mite allergens, and in other applications, such as those disclosed below. The products and processes of the present invention are advantageous because they enable the detection of anti Der HMW-map antibodies in fluids of animals and the inhibition of IgE or Des° HMW-map protein activity associated with disease.
One embodiment of the present invention is an isolated Derma.toplzagoides allergenic composition including: (a) 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-HCI, 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. Such Dermatophagoides allergenic composition is also referred to herein as a Der HMW-map composition. A suitable gel filtration column includes any gel filtration column capable of excluding proteins having a molecular weight between about 50 kD
and about 150 kD. A preferred gel filtration column includes, but is not limited to a Sephacryl S-100 column. A suitable anion exchange column includes any anion exchange column capable of binding to a protein having a pI of less than about pI 6. A
preferred anion exchange column includes, but is not limited to a Q-Sepharose column. As used herein, "stimulating a B lymphocyte response" refers to increasing a humoral immune response in an animal that is induced preferentially by a Der HMW-map of the present invention and involves the activity of a B lymphocyte in the animal. As used herein, "stimulating a T lymphocyte response" refers to increasing a cellular immune response in an animal that is induced preferentially by a Der HMW-map of the present invention and involves the activity of a T lymphocyte in the animal.
One embodiment of the present invention is an isolated protein that includes a Der HMW-map protein. It is to be noted that the term "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. As such, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protein has been purified. An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology, or can be produced by chemical synthesis.
As used herein, a Der HMW-map protein can be a full-length protein or any homolog of such a protein. As used herein, a protein can be a polypeptide or a peptide, as the terms are used by those of skill in the art. Preferably, a Des 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 Den HMW-map protein.
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. Preferably, 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. According to the present invention, a peptide comprising a B lymphocyte epitope can be from about 4 residues to about 50 residues in length, preferably from about 5 residues to about 20 residues in length. According to the present invention, a peptide comprising a T lymphocyte epitope can be from about 4 residues to about 20 residues in length, preferably from about 8 residues to about 16 residues in length.
A Der HMW-map protein of the present invention, including a homolog, can be identified in a straight-forward manner by the protein's ability to induce an allergic response to DeY HMW-map protein. Examples of Des 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 andlor 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.
Der HMW-map protein homologs can be the result of natural allelic variation or natural mutation. Der HMW-map protein homologs of the present invention can _g_ also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant nucleic acid techniques to effect random or targeted mutagenesis.
One embodiment of the present invention is a Der HMW-map gene that includes the nucleic acid sequence SEQ ID N0:14, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:19, SEQ ID N0:20 SEQ ID N0:22, SEQ ID N0:34, SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:42, SEQ ID N0:43, and SEQ ID N0:45 as well as the complements of any of these nucleic acid sequences.
These nucleic acid sequences are further described herein. For example, nucleic acid sequence SEQ ID N0:14 represents the deduced sequence of the coding strand of a cDNA (complementary DNA) denoted herein as Des HMW-map gene nucleic acid molecule nDerf981~s2, the production of which is disclosed in the Examples.
Nucleic acid molecule nDerf981~sz comprises an apparently full-length coding region.
The complement of SEQ ID N0:14 (represented herein by SEQ ID N0:16) refers to the nucleic acid sequence of the strand complementary to the strand,having SEQ ID
N0:14, which can easily be determined by those skilled in the art. Likewise, a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is complementary to (i.e., can form a double helix with) the strand for which the sequence is cited. It should be noted that since nucleic acid sequencing technology is not entirely error-free, SEQ ID
N0:14 (as well as other nucleic acid and protein sequences presented herein) represents an apparent nucleic acid sequence of the nucleic acid molecule encoding a Der~ HMW-map protein of the present invention.
In another embodiment, 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
N0:14 or SEQ ID N0:16, or any other Der HMW-map nucleic acid sequence cited herein. For example, an allelic variant of a Den HMW-map gene including SEQ ID
N0:14 or SEQ ID N0:16, is a gene that occurs at essentially the same locus (or loci) in the genome as the gene including SEQ ID N0:14 and SEQ ID N0:16, 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. alleles corresponding to, or of, cited nucleic acid sequences) usually encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants of genes or nucleic acid molecules can also comprise alterations in the 5' or 3' untranslated regions of the gene (e.g., in regulatory control regions), or can involve alternative splicing of a nascent transcript, thereby bringing alternative exons into juxtaposition. Allelic variants are well known to those skilled in the art and would be expected to occur naturally within a given dust mite such as Derm,atop72agoides, since the respective genomes are diploid, and sexual reproduction will result in the reassortment of alleles.
In one embodiment of the present invention, an isolated Der HMW-map protein is encoded by a nucleic acid molecule that hybridizes under stringent hybridization conditions to a gene encoding a Der HMW-map protein. The minimal size of a Der HMW-map protein of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridizing under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. The size of a nucleic acid molecule encoding such a protein is dependent on the nucleic acid composition and the percent homology between the Der HMW-map nucleic acid molecule and the complementary nucleic acid sequence. It can easily be understood that 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 nucleic acid molecule capable of forming a stable hybrid with a gene encoding a Der HMW-map protein is typically at least about 12 nucleotides to about 15 nucleotides in length if the nucleic acid molecule is GC-rich and at least about 15 to about 17 bases in length if it is AT-rich. The minimal size of a nucleic acid molecule used to encode a Der HMW-map protein homolog of the present invention is from about 12 to about 18 nucleotides in length, preferably about nucleotides, or about 15 nucleotides, or about 18 nucleotides in length. Thus, 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. There is no limit, other than a practical limit, on 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. Preferably, the preferred size of a protein encoded by a nucleic acid molecule of the present invention is a portion of the protein that induces an immune response which is about 30 amino acids, more preferably about 35 amino acids and even more preferably about 44 amino acids in length.
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° Clohirzg:
A Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et al., 1984, Arcal. Bioclzern. 138, 267-284. As explained in detail in the cited references, 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. 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 Tm, of a given nucleic acid molecule. As defined in the formula below, Tm is the temperature at which two complementary nucleic acid molecule strands will disassociate, assuming 100% complementarity between the two strands:
Tm 81.5°C + 16.6 log M + 0.41 (%G + C) - 500/n - 0.61 (%formamide).

For nucleic acid molecules smaller than about 50 nucleotides, hybrid stability is defined by the dissociation temperature (Td), which is defined as the temperature at which 50% of the duplexes dissociate. For these smaller molecules, the stability at a standard ionic strength is defined by the following equation:
Td=4(G+C)+2,(A+T).
A temperature of 5°C below Td is used to detect hybridization between perfectly matched molecules.
Also well known to those skilled in the art is how base-pair mismatch, i.e.
differences between two nucleic acid molecules being compared, including non-complementarity of bases at a given location, and gaps due to insertion or deletion of one or more bases at a given location on either of the nucleic acid molecules being compared, will affect Tm or Td for nucleic acid molecules of different sizes.
For example, Tm decreases about 1°C for each 1% of mismatched base-pairs for hybrids greater than about 150 bp, and Td decreases about 5°C for each mismatched base-pair for hybrids below about 50 bp. Conditions for hybrids between about 50 and about 150 base-pairs can be determined empirically and without undue experimentation using standard laboratory procedures well known to those skilled in the art.
These simple procedures allow one skilled in the art to set the hybridization conditions (by altering, for example, the salt concentration, the formamide concentration or the temperature) so that only nucleic acid hybrids with less than a specified %
base-pair mismatch will hybridize. Stringent hybridization conditions are commonly understood by those skilled in the art to be those experimental conditions that will allow hybridization between molecules having about 30% or less base-pair mismatch (i.e., about 70% or greater identity). Because one skilled in the art can easily determine whether a given nucleic acid molecule to be tested is less than or greater than about 50 nucleotides, and can therefore choose the appropriate formula for determining hybridization conditions, he or she can determine whether the nucleic acid molecule will hybridize with a given gene under stringent hybridization conditions and similarly whether the nucleic acid molecule will hybridize under conditions designed to allow a desired amount of base pair mismatch.
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. Examples of common hybridization reaction techniques include, but are not limited to, the well-known Southern and northern blotting procedures. Typically, 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.
For example, if the skilled artisan wished to identify a nucleic acid molecule that hybridizes under stringent hybridization conditions with a Dernzatophagoides farihae and/or Dermatophagoides ptero~zyssius nucleic acid molecule of about 150 by in length, the following conditions could preferably be used. The average G +
C
content of Dermatophagoides fari~aae and Dermatophagoides pterorayssius DNA is about 39%. The unknown nucleic acid molecules would be attached to a support membrane, and the 150 by 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 NaCI and about 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain the desired concentration of SSC. In order to achieve high stringency hybridization, the skilled artisan would calculate the washing conditions required to allow up to 30% base-pair mismatch. For example, in a wash solution comprising 1X SSC and 0% formamide, the Tm of perfect hybrids would be about 80°C:
81.5°C + 16.6 log (.15M) + (0.41 x 39) - (500/150) - (0.61 x 0) =
80.4°C.
Thus, to achieve hybridization with nucleic acid molecules having about 30%
base-pair mismatch, 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. For example, it is appreciated by one skilled in the art that as the nucleic acid molecule to be tested for hybridization against nucleic acid molecules of the present invention having sequences specified herein becomes longer than nucleotides, the Tm for a hybridization reaction allowing up to 30% base-pair mismatch will not vary significantly from 50°C.
Furthermore, it is known in the art that there are commercially available computer programs for determining the degree of similarity between two nucleic acid sequences. These computer programs include various known methods to determine the percentage identity and the number and length of gaps between hybrid nucleic acid molecules. Preferred methods to determine the percent identity among amino acid sequences and also among nucleic acid sequences include analysis using one or more of the commercially available computer programs designed to compare and analyze nucleic acid or amino acid sequences. These computer programs include, but are not limited to, GCGTM (available from Genetics Computer Group, Madison, WI), DNAsisTM (available from Hitachi Software, San Bruno, CA) and MacVectorTnn (available from the Eastman Kodak Company, New Haven, CT). A preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
One embodiment of the present invention includes Der HMW-map proteins.
In one embodiment, Der HMW-map proteins of the present invention include proteins that, when submitted to reducing 12% Tris glycine 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 fari~2ae mites using the method described in detail in Example 1. Preferably, 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 mapA
and mapB, the identification of which zs described in the Examples section.
In another embodiment, Der HMW-map proteins of the present invention include proteins that, when submitted to reducing 14% Tris glycine 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 Def-n2ataphagoides fariniae mites using the method described in detail in Example 9. Preferably, 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.
In another embodiment, 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 N0:16, SEQ ID N0:19, SEQ ID N0:22, SEQ ID
N0:36, SEQ m NO:39, SEQ ID N0:42, SEQ ID N0:45 and a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33 the complement thereof.
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 1X SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid sequence selected from the group consisting of SEQ ID N0:16, SEQ ll~
N0:19, SEQ ID N0:22, SEQ ID N0:36, SEQ ID N0:39, SEQ ID N0:42, SEQ ID N0:45, and a complement of a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33; 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 N0:14, SEQ ID
N0:17, SEQ ID N0:20, SEQ ID N0:34, SEQ ID N0:37, SEQ ID N0:40, SEQ ID
N0:43, and/or a complement of a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33;, also preferred are fragments of such proteins. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
Additional preferred Der HMW-map proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:l, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID
N0:9, SEQ ID N0:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID N0:13, SEQ ID
N0:15, SEQ ID N0:18, SEQ ID N0:21, SEQ ID N0:23, SEQ ID N0:24, SEQ ID
N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID
N0:35, SEQ ID NO:38, SEQ ID N0:41, SEQ ID N0:44, and proteins comprising homologs of a protein having the amino acid sequence SEQ TD NO:l, SEQ ID N0:2, SEQ ID N0:3, SEQ 117 N0:4, SEQ ZD N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID
N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID N0:11, SEQ ID N0:12, SEQ )D
N0:13, SEQ ID N0:15, SEQ ID N0:18, SEQ ID N0:21, SEQ ID N0:23, SEQ ID
N0:24, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ 117 N0:33, SEQ ID N0:35, SEQ ID N0:38, SEQ ID N0:41, SEQ ID N0:44 in which such a homolog comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ll~ NO:1, SEQ ID N0:2, SEQ ID
N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID
N0:9, SEQ ID N0:10, SEQ ID NO:l 1, SEQ ID N0:12, SEQ ID N0:13, SEQ D7 N0:15, SEQ ID N0:18, SEQ ID N0:21, SEQ ID N0:23, SEQ ID N0:24, SEQ ID
N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID
N0:35, SEQ ID N0:38, SEQ ID N0:41, SEQ ID N0:44 Likewise, also preferred are proteins encoded by nucleic acid molecules encoded by nucleic acid molecules having nucleic acid sequence SEQ ID N0:14, SEQ ID N0:17, SEQ ID N0:20, SEQ ID
N0:34, SEQ ID N0:37, SEQ ID N0:40, SEQ ID N0:43 andJor a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33, or by homologs thereof.
A preferred isolated protein of the present invention is a protein encoded by at least one of the following nucleic acid molecules: nDerf981~sz, nDerf981~~s, nDerf981sos~ ~e~98i6zn nDerp981sz~, nDerp9814~0, nDerf60s1o, 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 N0:14, SEQ ID
N0:17, SEQ ID N0:20, SEQ ID N0:34, SEQ ID N0:37, SEQ ID N0:40, SEQ ID N0:43; or a protein encoded by an allelic variant of any of these listed nucleic acid molecule.
Translation of SEQ ID N0:14, the coding strand of nDerf981~sz~ Yields a protein of about 555 amino acids, denoted herein as PDerf98sss, the amino acid sequence of which is presented in SEQ ID N0:15, assuming a first in-frame codon extending from nucleotide 1 to nucleotide 3 of SEQ 117 N0:14. The complementary strand of SEQ ID N0:14 is presented herein as SEQ ID N0:16. The amino acid sequence of PDerf98sss is encoded by the nucleic acid molecule nDerf981~~s, having a coding strand denoted SEQ 117 N0:17 and a complementary strand denoted SEQ ID
N0:19. Analysis of SEQ ID N0:15 suggests the presence of a signal peptide spanning from about amino acid 1 through about amino acid 19. The proposed mature protein, denoted herein as PDerf98s36, contains about 536 amino acids, the sequence of which is represented herein as SEQ ID N0:21, and is encoded by a nucleic acid molecule refeiTed to herein as nDerf9816os, represented by SEQ ID N0:20, the coding strand, and SEQ ID N0:22, the complementary strand.
Translation of SEQ ID NO:34, the coding strand of nDerp981~z~, Yields a protein of about 509 amino acids, denoted herein as PDerp98so9, the amino acid sequence of which is presented in SEQ ID NO:35, assuming a first in-frame codon extending from nucleotide 14 to nucleotide 16 of SEQ ID N0:34. The complementary strand of SEQ ID N0:34 is presented herein as SEQ ID N0:36. The amino acid sequence of PDerpf98so9 is encoded by the nucleic acid molecule nDerp981sz~, having a coding strand denoted SEQ ID NO:37 and a complementary strand denoted SEQ ID
N0:39. Analysis of SEQ ID N0:35 suggests the presence of a signal peptide spanning from about amino acid 1 through about amino acid 19. The proposed mature protein, denoted herein as PDerp9849o, contains about 490 amino acids, the sequence of which is represented herein as SEQ ID N0:41, and is encoded by a nucleic acid molecule referred to herein as nDerp9814~0, represented by SEQ ID N0:40, the coding strand, and SEQ ID N0:42, the complementary strand.
Translation of SEQ ID N0:43, the coding strand of nDerf60sio, a nucleic acid molecule encoding a portion of the D. fari~ae 60-kD antigen protein yields a protein of about 170 amino acids, denoted herein as PDerf601~o, the amino acid sequence of which is presented as SEQ ID N0:44, assuming a first in-frame codon extending from nucleotide 1 to nucleotide 3 of SEQ ID N0:43. The complementary sequence to SEQ
ID NO:43 is presented herein as SEQ ID N0:45.
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 PDerf98sss. More preferred is a Der HMW-map protein comprising PDerf98sss, PDerf98ssG , PDerp98so9, PDerp98ø9o, and/or PDerf601~o; and proteins encoded by allelic variants of nucleic acid molecules encoding proteins PDerf98sss~
PDerf98s3~ , PDerp98so9, PDerp98ø9o, and/or PDerf601~o.
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 N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ
ID N0:12, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:18, SEQ ID N0:21, SEQ ID
N0:23, SEQ ID N0:24, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID
N0:32, SEQ ID N0:33, SEQ ID N0:35, SEQ ID N0:38, SEQ ID N0:41, and/or SEQ

ID N0:44. More preferred are Der HMW-map proteins comprising amino acid sequences SEQ ID NO:l, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID N0:10, SEQ ID
NO:l 1, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:18, SEQ ID
N0:21, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:29, SEQ ID N0:30, SEQ ID
NO:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:35, SEQ ID N0:38, SEQ ID
N0:41, and/or SEQ ID N0:44; and Der HMW-map proteins encoded by allelic variants of nucleic acid molecules encoding Der HMW-map proteins having amino acid sequences SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID
N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:l 1, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:18, SEQ ID N0:21, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:29, SEQ ID NO:30, SEQ ID N0:31, SEQ ID N0:32, SEQ 117 N0:33, SEQ ID N0:35, SEQ ll~ N0:38, SEQ ID N0:41, and/or SEQ ID N0:44.
In one embodiment of the present invention, Der HMW-map proteins comprise amino acid sequence SEQ ID N0:15, SEQ ID N0:35, and/or SEQ ID N0:44 (including, but not limited to, the proteins consisting of amino acid sequence SEQ ID
N0:15, SEQ ID N0:35, and/or SEQ ID N0:44, 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 N0:15, SEQ ID
N0:35, and/or SEQ ID N0:44.
In one embodiment, a preferred Der HMW-map protein comprises an amino acid sequence of 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, mode preferably at least about 200 amino acids in length, even more preferably at least about 250 amino acids in length. Within this embodiment, a preferred Der HMW-map protein of the present invention has an amino acid sequence comprising at least a portion of SEQ ID N0:15. In another embodiment, a preferred Der HMW-map protein comprises a full-length protein, i.e., a protein encoded by a full-length coding region.

Additional preferred Der HMW-map proteins of the present invention include proteins encoded by nucleic acid molecules comprising at least a portion of nDerf981~sz, nDerf98166s, nDerf9816o8, nDerp9816zv nDerp981sz~, nDerp981a~o, and nDerf60slo, as well as Der HMW-map proteins encoded by allelic variants of such nucleic acid molecules.
Also preferred are Der HMW-map proteins encoded by nucleic acid molecules having nucleic acid sequences comprising at least a portion of SEQ ID N0:14, SEQ
ID N0:17, SEQ ID N0:20, SEQ ID N0:34, SEQ ID N0:37, SEQ ID N0:40 SEQ ID
N0:43 and/or a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33, as well as allelic variants of these nucleic acid molecules.
In another embodiment, a preferred Der HMW-map protein of the present invention is encoded by a nucleic acid molecule comprising at least about 12 nucleotides, preferably at least about 16 nucleotides, more preferably at least about 18 nucleotides, more preferably at least about 20 nucleotides, more preferably at least about 25 nucleotides, more preferably at least about 50 nucleotides, more preferably at least about 100 nucleotides, more preferably at least about 350 nucleotides, more preferably at least about 450 nucleotides, more preferably at least about 500 nucleotides, and even more preferably at least about 800 nucleotides. Within this embodiment is a Der HMW-map protein encoded by at least a portion nDerf981~sz~
nDerp9816zv and/or nDerf60slo or by an allelic variant of these nucleic acid molecules.
In yet another embodiment, 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.
One embodiment of 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 aDer HMW-map protein, reduce an IgE response against a Der HMW-map protein; andlor assist purification of a Def° 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). Fusion segments can be joined to amino and/or carboxyl termini of the Der HMW-map protein-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of a Der HMW-map protein. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of a Der HMW-map protein-containing domain.
Preferred fusion segments include a metal binding domain (e.g., a poly-histidine segment); an immunoglobulin binding domain (e.g., Protein A; Protein G; T
cell; B
cell; Fc receptor or complement protein antibody-binding domains); a sugar binding domain (e.g., a maltose binding domain); a "tag" domain (e.g., at least a portion of -galactosidase, a strep tag peptide, other domains that can be purified using compounds that bind to the, domain, such as monoclonal antibodies); and/or a linker and enzyme domain (e.g., alkaline phosphatase domain connected to a Der HMW-map protein by a linker). More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag peptide, such as that available from Biometra in Tampa, FL; and a phage T7 S 10 peptide.
In another embodiment, a Der HMW-map protein of the present invention also includes at least one additional protein segment that is capable of desensitizing an animal from one or more allergens. 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.
Examples of 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, Derznatophagoides, Alterrzaria, Aspergillus, Cladospoz~ium, Fusariuzn, Helmizzthosporium, Mucor, Pezzicillium, Pullulaz°ia, Rlzizopus and/or Tz~icoplzytozz;
parasite allergens from helminths; or ectoparasite allergens from arachnids, insects and leeches, including fleas, ticks, flies, mosquitos, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting gnats, ants, spiders, lice; mites and true bugs.
The present invention also includes mimetopes of a Der HMW-map protein of the present invention. As used herein, a mimetope of a Der HMW-map protein of the present invention refers to any compound that is able to mimic the activity of such a Der HMW-map protein (e.g., ability to bind to induce an immune response against Der HMW-map protein), often because the mimetope has a structure that mimics the Dez~ HMW-map protein. It is to be noted, however, that the mimetope need not have a structure similar to a Der HMW-map protein as long as the mimetope functionally mimics the protein. 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.
Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner, (e.g., an anti-Der HMW-map protein antibody). A mimetope can also be obtained by, for example, rational drug design. In a rational drug design procedure, the three-dimensional structure of a compound of the present invention can be analyzed by, for example, nuclear magnetic resonance (NMR) or x-ray crystallography. The three-dimensional structure can then be used to predict structures of potential mimetopes by, for example, computer modeling. The predicted mimetope structures can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a mimetope from a natural source. Specific examples of Der HMW-map protein mimetopes include anti-idiotypic antibodies, oligonucleotides produced using SelexTM technology, peptides identified by random screening of peptide libraries and proteins identified by phage display technology. A preferred mimetope is a peptidomimetic compound that is structurally and/or functionally similar to a Der HMW-map protein of the present invention, particularly to an epitope of Der HMW-map protein that induces an immune response.
The present invention also includes muteins of a Der~ HMW-map protein of the present invention. As used herein, a mutein refers to a particular homolog of a Der 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 then apeutic 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. Preferably, 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.

In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subjected to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. An isolated Der HMW-map nucleic acid molecule of the present invention, or a homolog thereof, can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis. 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.
A Der HMW-map nucleic acid molecule homolog can be produced using a number of methods known to those skilled in the art, see, for example, Sambrook et al., 1989, Molecular CloT2ing: A Laboratory Manual, Cold Spring Harbor Labs Press;
Sambrook et al., ibid. For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques such as site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, PCR
amplification, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof. 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 Def- HMW-map protein of the present invention, examples of such proteins being disclosed herein. Although the phrase "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 Def° 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. As will be disclosed in more detail below, such 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. In additional embodiments, a nucleic acid molecule of the present invention can encode a desensitizing protein (e.g., a Def~ HMW-map protein of the present invention), the nucleic acid molecule being delivered to the animal, for example, by direct injection (i.e, as a DNA reagent) or in a vehicle such as a recombinant virus reagent or a recombinant cell reagent.
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:1, SEQ D7 N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID
N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:18, SEQ ID N0:21, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:35, SEQ ID N0:38, SEQ ID N0:41, and/or SEQ ID
N0:44. 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 N0:2, SEQ ID NO:3, SEQ ID
N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID N0:10, SEQ m NO:11, SEQ ID N0:12, SEQ ll~ N0:13, SEQ ID N0:15, SEQ ID N0:18, SEQ ID N0:21, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:35, SEQ ID N0:38, SEQ ID N0:41, and/or SEQ 117 N0:44.
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 1X SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid sequence selected from the group consisting of SEQ ID N0:14, SEQ ID
N0:16, SEQ ID N0:17, SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:22, SEQ m N0:34, SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:39, SEQ ID N0:40, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:45 and/or a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33 and a complement thereof.
The present invention also includes fragments of any nucleic acid molecule disclosed herein. According to the present invention, 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. For example, 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.
In one embodiment of the present invention, a preferred DeY 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
N0:14, SEQ ID N0:16, SEQ D7 N0:17, SEQ ID N0:19, SEQ ID N0:20, SEQ ID
N0:22, SEQ ID N0:34, SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:40, SEQ ID
N0:42, SEQ ID N0:43, SEQ ID N0:45, and a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33 and a complement thereof.
Another embodiment of the present invention includes a nucleic acid molecule comprising at least about 150 base-pairs, wherein the nucleic acid molecule hybridizes, in a solution comprising 1X SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid sequence selected from the group consisting of SEQ ID
N0:14, SEQ m N0:16, SEQ ID N0:17, SEQ ID N0:19, SEQ ID N0:20, SEQ ID
N0:22, SEQ m N0:34, SEQ ID N0:36, SEQ m N0:37, SEQ ID N0:39, SEQ ID
N0:40, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:45, and/or a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33 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 1X SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid sequence selected from the group consisting of SEQ ID N0:14, SEQ ID
N0:16, SEQ m N0:17, SEQ ID N0:19, SEQ ID N0:20, SEQ ID NO:22, SEQ ID N0:34, SEQ m N0:36, SEQ ID N0:37, SEQ ID N0:39, SEQ ID NO:40, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:45 and a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33 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 _27_ 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 N0:14, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:19, SEQ
ID N0:20, SEQ ID N0:22, SEQ ID N0:34, SEQ ID N0:36, SEQ m N0:37, SEQ ID
N0:39, SEQ ID N0:40, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:45, and a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID
N0:33 and a complement thereof. Also preferred are fragments of any of such nucleic acid molecules. Percent identity may be determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
One embodiment of the present invention is a nucleic acid molecule comprising all or part of nucleic acid molecules nDerf981~s2, nDerf981~6s and nDerf9816o8, nDerp9816av nDerp981sz~, nDerp981~~0, and/or nDerf60slo, or allelic variants of these nucleic acid molecules. Another preferred nucleic acid molecule of the present invention includes at least a portion of nucleic acid sequence SEQ
ID
N0:14, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:19, SEQ ID N0:20, SEQ ID
N0:22, SEQ ID N0:34, SEQ ID N0:36, SEQ ID N0:37, SEQ ID N0:39, SEQ ID
N0:40, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:45 and/or a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33, 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:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID
N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:1 l, SEQ ID N0:12, SEQ ID N0:13, SEQ >D N0:15, SEQ ID N0:18 , SEQ ID N0:21, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ >D N0:35, SEQ ID N0:38, SEQ ID N0:41, SEQ >D N0:41, and/or SEQ m N0:44. Such 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.
In one embodiment, a Der HMW-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 PDerf98sss, PDerp98so9, and/or PDerf601~o.
Even more preferred is a nucleic acid molecule encoding PDerf98sss, PDerf98s3~, PDerp98so9, PDerp9849o, and/or PDerf601~o, and/or an allelic variant of such nucleic acid molecules.
In another embodiment, a Der HMW-map nucleic acid molecule of the present invention encodes a protein having an amino acid sequence 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 SEQ
ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:l l, SEQ
ID N0:12, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:18 , SEQ ID N0:21, SEQ ID
N0:23, SEQ 117 N0:24, SEQ ID N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID
N0:32, SEQ ID N0:33, SEQ ID N0:35, SEQ ID N0:38, SEQ ID N0:41, SEQ TD
N0:41, and/or SEQ ID NO:44. The present invention also includes a Den HMW-map nucleic acid molecule encoding a protein having at least a portion of SEQ ID
NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ll~ N0:6, SEQ ID
N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ DJ N0:10, SEQ ID NO:1 l, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:18 , SEQ ID NO:21, SEQ ID N0:23, SEQ D7 N0:24, SEQ m N0:29, SEQ ID N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ 117 N0:33, SEQ 117 N0:35, SEQ ID N0:38, SEQ ID N0:41, SEQ ID N0:41, and/or SEQ ID N0:44, as well as allelic variants of a Der HMW-map nucleic acid molecule encoding a protein having these sequences, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.
In another embodiment, a preferred Def~ HMW-map nucleic acid molecule encodes a Der 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.
Knowing the nucleic acid sequences of certain Der- HMW-map nucleic acid molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain other Der HMW-map nucleic acid molecules.
Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention;
traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries; and PCR amplification of appropriate libraries or DNA
using oligonucleotide primers of the present invention. A preferred library to screen or from Which to amplify nucleic acid molecules includes a Dermatophagoides farihae and/or Dermatophagoides ptero~yssius library, such as the libraries disclosed herein in the Examples. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.
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. A preferred oligonucleotide of the present invention has a maximum size of preferably about 200 nucleotides, more preferably about 150 nucleotides and even more preferably about 100 nucleotides. The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules.
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. Such 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 molecules) 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 Den HMW-map nucleic acid molecules of the present invention.
One type of recombinant vector, referred to herein as 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. As used herein, 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. Preferably, 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.

In particular, 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. In particular, 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.
Preferred transcription control sequences include those which function in bacterial, yeast, insect and mammalian cells, such as, but not limited to, tac, lac, try, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda pL and lambda pR and fusions that include such promoters), bacteriophage T7, T7la.c, bacteriophage T3, bacteriophage SP6, bacteriophage SPO1, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).
Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with canines or felines.
Suitable and preferred nucleic acid molecules to include in recombinant vectors of the present invention are as disclosed herein. Preferred nucleic acid molecules to include in recombinant vectors, and particularly in recombinant molecules, include nDerf981~sz, nDerf981sss nDerf9816os, nDerp98,~zi, nDerp981sz.,, nDerp981a~o, and nDerf60s io.
Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed Der HMW-map protein of the present invention to be secreted from the cell that produces the protein and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Examples 'of suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention. Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments, as well as natural signal segments. Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein. In addition, a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment. Recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A
recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained. Preferred nucleic acid molecules with which to transform a cell include Der HMW-map nucleic acid molecules disclosed herein.
Particularly.preferred nucleic acid molecules with which to transform a cell include nDerf981~sz, nDerf98166s nDerf9816os, nDerp9816zn nDerp981s2~, nDerp981~~0, and nDerf60s lo.
Suitable host cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of multivalent vaccines). Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing Def HMW-map proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), other insect, other animal and plant cells. Preferred host cells include bacterial, mycobacterial, yeast, parasite, insect and mammalian cells. More preferred host cells include Sal~io~zella, Escherichia, Bacillus, Listeria, Saccl2as~o~zyces, Spodoptera, Mycobacteria, Trichoplusia, 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. coli K-12 derivatives; Salmonella typl2i; Sa.lf~2o~zella typhimuf°ium, including attenuated strains such as UK-1 X3987 and SR-11 x4072; Spodoptera frugiperda; Ti~ichoplusia ni;
BHK
cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK31 cells and/or HeLa cells.
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.
A recombinant molecule of the present invention is a molecule that can include at least one of any nucleic acid molecule heretofore described operatively linked to at least one of any transcription control sequence capable of effectively regulating expression of the nucleic acid molecules) in the cell to be transformed, examples of which are disclosed herein.
A recombinant cell of the present invention includes any cell transformed with at least one of any Der HMW-map nucleic acid molecule of the present invention.
Suitable and preferred Der HMW-map nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transform cells are disclosed herein.
Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications.
Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of 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. In one embodiment, 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. An effective medium refers to any medium in which a cell is cultured to produce a Der HMW-map protein of the present invention. Such a medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins.
Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell.
Such culturing conditions are within the expertise of one of ordinary skill in the art.
Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or be retained on the outer surface of a cell or viral membrane. The phrase "recovering the protein", as well as similar phrases, refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification.
Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatogr aphy, 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 Des HMW-map protein of the present invention or a mimetope thereof (i.e., anti-Der HMW-map protein antibodies).
As used herein, the term "selectively binds to" a Der 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-Den HMW-map protein antibody preferably selectively binds to a portion of a Der HMW-map protein that induces an immune response in an animal.
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. In another method, 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. For example, 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 Der HMW-map protein of the present invention can be contained in a formulation, herein referred to as a Der HMW-map protein formulation. For example, 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. Examples of suitable buffers include any buffer in which a Der HMW-map protein can function to selectively bind to an antibody that specifically binds to Der HMW-map protein, such as, but not limited to, phosphate buffered saline, water, saline, phosphate buffer, bicarbonate buffer, HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffered saline), TES buffer (Tris-EDTA buffered saline), Tris buffer and TAE buffer (Tris-acetate-EDTA).
Examples of carriers include, but are not limited to, polymeric matrices, toxoids, and serum albumins, such as bovine serum albumin. 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 DeY
HMW-map protein of the present invention.
In addition, 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. As used herein, an antigen refers to any molecule capable of being selectively bound by an antibody. As used herein, an allergen refers to any .antigen that is capable of stimulating production of antibodies involved in an allergic response in an animal. As used herein, 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.
In accordance with the present invention, virtually any substance can act as an antigen and elicit an antibody response, i.e., can function as an epitope. For example, antibodies can be raised in response to carbohydrate epitopes, including saecharides and/or polysaccharides that are attached to a protein, a so-called glycosylated protein.
However, a saccharide and/or polysaccharide may act as an antigen alone, without a protein being present. The terminal sugar of a carbohydrate moiety, as well as internal sugars can serve as an epitope. Polysaccharide may be present as a branched chain, in which case epitopes may comprise sugars that are not contiguous in sequence, but are adjacent spatially. Unusual, insect-specific sugars, not normally seen in mammalian proteins, may be present on glycoprotein derived from insect nucleic acid molecules, and these unusual sugars can comprise an epitope recognized by a mammalian immune system.
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. Such an epitope, referred to herein as 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. farihae 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 an epitope can be, but need not be, joined to a carrier or other molecule. ADer NP epitope has at least one of the following identifying characteristics: (a) the epitope is resistant to (3-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 preferred Der NP epitope has,all such identifying characteristics. ADer NP epitope can selectively bind to IgE
of dogs or cats that are allergic to mites. While not being bound by theory, it is believed that a Der NP epitope comprises a carbohydrate moiety that apparently does not include an N-linked glycan. Identification of the structural characteristics of such an epitope can be determined by one skilled in the art. In one embodiment, there is provided an isolated antibody that selectively binds to a Def° NP
epitope. The present invention also includes a derivative of a Der NP epitope, i.e., a compound that mimics the activity of such an epitope (e.g. is a Der NP epitope mimetope) and is capable of binding to antibody raised against a native (i.e. seen in nature) Der NP
epitope.
A reagent comprising a Der NP epitope of the present invention can be used in a variety of ways in accordance with the present invention. Such a reagent can be a desensitizing compound or a detection reagent to test for mite allergy susceptibility or sensitivity. In one embodiment, a therapeutic composition of the present invention includes a reagent comprising a Der NP epitope. In another embodiment, an assay kit of the present invention includes a reagent comprising a Der NP epitope. One embodiment of the present invention is a method to identify an animal susceptible to or having an allergic response to a mite. 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.
One embodiment of the present invention is an ih vivo test that is capable of detecting whether an animal is hypersensitive to Der HMW-map protein. An iiz vivo hypersensitivity test of the present invention is particularly useful for identifying animals susceptible to or having allergy to mite allergens. A- suitable ih vivo hypersensitivity test of the present invention can be, but is not limited to, a skin test comprising administering (e.g., intradermally injecting or superficial scratching) an effective amount of a formulation containing Der HMW-map protein, or a mimetope thereof. Methods to conduct skin tests of the present invention are known to those of skill in the art and are briefly disclosed herein.
Suitable formulations to use in an irc vivo skin test include Der HMW-map protein, homologs of Der HMW-map protein and/or mimetopes of Der HMW-map protein.
It is understood by one of skill in the art that a suitable amount of Der HMW-map protein formulation for use in a skin test of the present invention can vary widely depending on the allergenicity of the formulation used in the test and on the site at which the product is delivered. Suitable amounts of Def° HMW-map protein formulation for use in a skin test of the present invention include an amount capable of forming reaction, such as a detectable wheal or induration (hardness) resulting from an allergic reaction to the formulation. 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 (fig) to about 100 fig, more preferably from about 1 x 10-' ~g to about 10 fig, and even more preferably from about 1 x 10-~ ~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.
According to the present invention, Dej- HMW-map protein of the present invention can be combined with an immunopotentiator (e.g., carriers or adjuvants of the present invention as defined in detail below). A novel aspect, however, of the present invention is that DeY HMW-map protein of the present invention can induce a hypersensitive response in the absence of an immunopotentiator, particularly in canines.
A skin test of the present invention further comprises administering a control solution to an animal. A control solution can include a negative control solution and/or a positive control solution. A positive control solution of the present invention contains an effective amount of at least one compound known to induce a hypersensitive response when administered to an animal. A preferred compound for use as positive control solution includes, but is not limited to, histamine. A
negative control solution of the present invention can comprise a solution that is known not to induce a hypersensitive response when administered to an animal. As such, a negative control solution can comprise a solution having compounds essentially incapable of inducing a hypersensitive response or simply a buffer used to prepare the formulation, such as saline. An example of a preferred negative control solution is 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 samples) and control samples) into an animal and comparing the reactions to the experimental samples) 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 _q.2_ 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.
As such, if an experimental sample produces a reaction greater than or equal to the size of a wheat produced by administration of a positive control sample to an animal, then that animal is hypersensitive to the experimental sample. Conversely, if 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.
Preferred wheat sizes for evaluation of the hypersensitivity of an animal range from about 16 mm to about 8 mm, more preferably from about 15 mm to about 9 mm, and even more preferably from about 14 mm to about 10 mm in diameter.
Preferably, the ability or inability of an animal to exhibit an immediate hypersensitive response to a formulation of the present invention is determined by measuring wheat 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.
Preferably, 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 andlor 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 administr ation during the time periods defined directly above.
In a preferred embodiment, 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 Def-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. As used herein, 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. As used herein, feline refers to any member of the cat family, including domestic cats, wild cats and zoo cats. Examples of cats include, but are not limited to, domestic cats, lions, tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs and servals. As used herein, 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 a Der 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 aDer 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.
As used herein, the term "contacting" refers to combining or mixing, in this case a putative antibody-containing composition with a Der HMW-map protein.
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). As used herein, the term selectively binds to an antibody refers to the ability of a Def~ 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.
As used herein, the term "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.
In one embodiment, 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. 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 andlor 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. In another embodiment, a composition includes collected bodily fluid that is pretreated to concentrate immunoglobulin contained in the fluid. For example, immunoglobulin contained in a bodily fluid can be precipitated from other proteins using ammonium sulfate. A
preferred composition of the present method is serum.
In another embodiment, an antibody-containing composition of the present method includes a cell that produces IgE or IgG. Such 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). Such 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.
In other assays, conjugation (i.e., attachment) of a detectable marker to the Def°
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. Examples of 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 ImmunoPureO NeutrAvidin available from Pierce, Rockford, IL).
In one embodiment, 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 Der 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.
In another embodiment, a Der HMW-map protein: antibody complex is detected by contacting a putative antibody-containing composition with a Der HMW-map protein and then contacting the complex with an indicator molecule.
Suitable indicator molecules of the present invention include molecules that can bind to either the Der HMW-map protein or to the antibody bound to the Der HMW-map protein.
As such, an indicator molecule can comprise, for example, an antigen and an antibody, depending upon which portion of the Der HMW-map protein: antibody complex is being detected. Preferred indicator molecules that are antibodies include, for example, anti-IgE antibodies, anti-IgG antibodies and antibodies that are known bind to Der HMW-map protein but bind to a different epitope on Der HMW-map protein than antibodies identified in the putative antibody-containing composition.
Preferred lectins include those lectins that bind to high-mannose groups. An indicator molecule itself can be attached to a detectable marker of the present invention. For example, an antibody can be conjugated to biotin, horseradish peroxidase, alkaline phosphatase or fluorescein.
In one preferred embodiment, aDer 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. Examples of such an anti-IgE or an anti-IgG
antibody include, but are not limited to, a secondary antibody that is an anti-isotype antibody (e.g., an antibody that selectively binds to the constant region of an IgE or an IgG), an antibody-binding bacterial surface protein (e.g., Protein A or Protein G), an antibody-binding cell (e.g., a B cell, a T cell, a natural killer cell, a polymorphonuclear leukocyte cell, a monocyte cell or a macrophage cell), an antibody-binding eukaryotic cell surface protein (e.g., a Fc receptor), and an antibody-binding complement protein.
Preferred indicator molecules include, but are not limited to, an anti-feline IgE
antibody, an anti-feline IgG antibody, an anti-canine IgE antibody, an anti-canine IgG
antibody, an anti-human IgE antibody, and an anti-human IgG antibody. As used herein, an anti-IgE or anti-IgG antibody includes not only a complete antibody but also any subunit or portion thereof that is capable of selectively binding to an IgE or IgG
heavy chain constant region. For example, an anti-IgE reagent or anti-IgG
reagent can include an Fab fragment or a Flab' )Z fragment, both of which are described in detail in Janeway et al., in Immm2obiology, the Immune Systen-a i~ Health. and Disease, Garland Publishing, Inc., NY, 1996.
In another preferred embodiment, aDef~ HMW-map protein:antibody complex is detected by contacting the complex with an indicator molecule that selectively binds to Der HMW-map protein at a different epitope than the epitope at which an antibody in a putative antibody-containing composition binds to Der HMW-map protein.
In one embodiment a complex can be formed and detected in solution. In another embodiment, a complex can be formed in which one or more members of the complex are immobilized on (e.g., coated onto) a substrate. Immobilization techniques are known to those skilled in the art. Suitable substrate materials include, but are not limited to, plastic, glass, gel, celluloid, paper, PVDF (poly-vinylidene-fluoride), nylon, nitrocellulose, and particulate materials such as latex, polystyrene, nylon, nitrocellulose, agarose and magnetic resin. 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. In one embodiment, 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. As such, 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. As such, an indicator molecule preferably is not immobilized to the same substrate as a capture molecule prior to exposure of the capture molecule to a putative IgE-containing composition or a putative IgG-containing composition.
A preferred immunoabsorbent assay method includes a step of either:
(a) immobilizing a Der HMW-map protein on a substrate prior to contacting a Der HMW-map protein with a putative IgE-containing composition or a putative IgG-containing composition to form a Der HMW-map protein -immobilized substrate;
and (b) binding a putative IgE-containing composition or a putative IgG-containing composition on a substrate prior to contacting Der HMW-map protein with a putative IgE-containing composition or a putative IgG-containing composition, to form a putative IgE-containing composition-bound substrate or a putative IgG-containing composition-bound substrate, respectively. Preferably, the substrate includes a non-coated substrate, a Der HMW-map protein -immobilized substrate, an anti-IgE
antibody-immobilized substrate or anti-IgG antibody-immobilized substrate.
Both a capture molecule and an indicator molecule of the present invention are capable of binding to an IgE, an IgG or Der HMW-map protein. Preferably, a capture molecule binds to a different region of an IgE, an IgG or Der HMW-map protein than an indicator molecule, thereby allowing a capture molecule to be bound to an IgE, an IgG or Der HMW-map protein at the same time as an indicator molecule. The use of a reagent as a capture molecule or an indicator molecule depends upon whether the molecule is immobilized to a substrate when the molecule is exposed to an IgE, an IgG
or Der HMW-map protein. For example, a Der HMW-map protein of the present invention is used as a capture molecule when the Der HMW-map protein is bound on a substrate. Alternatively, a Der HMW-map protein is used as an indicator molecule when the Der HMW-map protein is not bound on a substrate. Suitable molecules for use as capture molecules or indicator molecules include, but are not limited to, a Der HMW-map protein of the present invention, an anti-IgE antibody reagent or an anti-IgG antibody reagent of the present invention.
An immunoabsorbent assay of the present invention can further comprise one or more layers and/or types of secondary molecules or other binding molecules capable of detecting the presence of an indicator molecule. For example, an untagged (i.e., not conjugated to a detectable marker) secondary antibody that selectively binds to an indicator molecule can be bound to a tagged (i.e., conjugated to a detectable marker) tertiary antibody that selectively binds to the secondary antibody.
Suitable secondary antibodies, tertiary antibodies and other secondary or tertiary molecules can be selected by those of skill in the art. Preferred secondary molecules of the present invention include an antigen, an anti-IgE idiotypic antibody (i.e., an antibody that binds to an epitope unique to the anti-IgE antibody), an anti-IgE isotypic antibody, an anti-IgG idiotypic antibody (i.e:, an antibody that binds to an epitope unique to the anti-IgG antibody), and an anti-IgG isotypic antibody. Preferred tertiary molecules can be selected by a skilled artisan based upon the characteristics of the secondary molecule. The same strategy is applied for subsequent layers.
In one embodiment, Def° HMW-map protein is used as a capture molecule by being immobilized on a substrate, such as a microtiter dish well or a dipstick. A
biological sample collected from an animal is applied to the substrate and incubated under conditions suitable (i.e., sufficient) to allow for Dey- HMW-map protein:antibody complex formation bound to the substrate (i.e., IgE or IgG in a sample binds to Der HMW-map protein immobilized on a substrate). Excess non-bound material (i.e., material from the biological sample that has not bound to the Der-HMW-map protein), if any, is removed from the substrate under conditions that retain antigen:antibody complex binding to the substrate. Preferred conditions are generally disclosed in Sambrook et al., ibid. An indicator molecule that can selectively bind to an IgE or an IgG bound to the antigen is added to the substrate and incubated to allow formation of a complex between the indicator molecule and the Der HMW-map protein:antibody complex. Excess indicator molecule is removed, a developing agent is added if required, and the substrate is submitted to a detection device for analysis.
A preferred indicator molecule for this embodiment is an anti-IgG antibody to detect IgG antibody bound to Der HMW-map protein or an anti-IgE antibody to detect IgE
antibody bound to Der HMW-map protein. Preferably the anti-IgG or anti-IgE
antibody are conjugated to biotin, to a fluorescent label or to an enzyme label.
In one embodiment, an anti-IgE or anti-IgG antibody (e.g., isotype or idiotype specific antibody) is used as a capture molecule by being immobilized on a substrate, such as a microtiter dish well or a dipstick. A biological sample collected from an animal is applied to the substrate and incubated under conditions suitable to allow for anti-IgE antibody:IgE complex or anti-IgG antibody:IgG complex formation, respectively, bound to the substrate. 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. Preferably, 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.
In one embodiment, an immunoabsorbent assay of the present invention does not utilize a capture molecule. In this embodiment, 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. Preferably, the Der HMW-map protein is conjugated to a detectable marker (preferably to biotin, an enzyme label or a fluorescent label). Excess DeY HMW-map protein is removed, a developing agent is added if required, and the substrate is submitted to a detection device for analysis.
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. 5,415,994, issued May 16, 1995, by Imrich et al; WO
94/29696, published December 22, 1994, by Miller et al.; and WO 94/01775, published January 20, 1994, by Pawlak et al. In one embodiment, a biological sample is placed in a lateral flow apparatus that includes the following components:
(a) a support structure defining a flow path; (b) a labeling reagent comprising a bead conjugated to Der HMW-map protein, the labeling reagent being impregnated within the support structure in a labeling zone; and (c) a capture reagent comprising an IgE-binding or an IgG-binding composition. The capture reagent is located downstream of the labeling reagent within a capture zone fluidly connected to the labeling zone in such a manner that the labeling reagent can flow from the labeling zone into the capture zone. The support structure comprises a material that does not impede the flow of the beads from the labeling zone to the capture zone. Suitable materials for use as a support structure include ionic (i.e:, anionic or cationic) material.
Examples of such a material include, but are not limited to, nitrocellulose (NC), PVDF, carboxymethylcellulose (CM). The support structure defines a flow path that is lateral and is divided into zones, namely a labeling zone and a capture zone. The apparatus can further comprise a sample receiving zone located along the flow path, more preferably upstream of the labeling reagent. The flow path in the support structure is created by contacting a portion of the support structure downstream of the capture zone, preferably at the end of the flow path, to an absorbent capable of absorbing excess liquid from the labeling and capture zones.
In this embodiment, the biological sample is applied to the sample receiving zone which includes a portion of the support structure. The labeling zone receives the sample from the sample receiving zone which is directed downstream by the flow path. The labeling zone comprises the labeling reagent that binds to IgE or IgG, or both. A preferred labeling reagent is Der HMW-map protein conjugated, either directly or through a linker, to a plastic bead substrate, such as to a latex bead. The substrate also includes a detectable marker, preferably a colorimetric marker.
Typically, the labeling reagent is impregnated to the support structure by drying or lyophilization. The sample structure also comprises a capture zone downstream of the labeling zone. 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 DeY 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.
In another embodiment, a lateral flow apparatus used to detect IgE or IgG
includes: (a) a support structure defining a flow path; (b) a labeling reagent comprising an anti-IgE or an anti-IgG antibody, or both, as described above, the labeling reagent impregnated within the support structure in a labeling zone; and (c) a capture reagent comprising Der HMW-map protein, the capture reagent being located downstream of the labeling reagent within a capture zone fluidly connected to the labeling zone in such a manner that the labeling reagent can flow from the labeling zone into the capture zone. The apparatus preferably also includes a sample receiving zone located along the flow path, preferably upstream of the labeling reagent. The apparatus preferably also includes an absorbent located at the end of the flow path.
An animal hypersensitive to Der HMW-map protein is identified by comparing the level of immunocomplex formation using samples of body fluid with the level of immunocomplex formation using control samples. An immunocomplex refers to a complex comprising an antibody and Der HMW-map protein (i.e., Der HMW-map protein:antibody complex). As such, immunocomplexes form using positive control samples and do not form using negative control samples. As such, if a body fluid sample results in immunocomplex formation greater than or equal to immunocomplex formation using a positive control sample, then the animal from which the fluid was taken is hypersensitive to the Der HMW-map protein bound to the substrate.

Conversely, if a body fluid sample results in immunocomplex formation similar to immunocomplex formation using a negative control sample, then the animal from which the fluid was taken is not hypersensitive to the Der HMW-map protein bound to the substrate.
It is within the scope of the present invention that two or more different skin tests and/or ih vitro tests can be used in combination for diagnostic purposes. For example, the immediate hypersensitivity of an animal to Der HMW-map protein can be tested using an iya vitro immunoabsorbent test capable of detecting IgE
antibodies specific for Der HMW-map protein in the animal's bodily fluid. While most animals that display delayed hypersensitivity to Der HMW-map protein also display immediate hypersensitivity to the allergen, a small number of animals that display delayed hypersensitivity to an allergen do not display immediate hypersensitivity to the allergen. In such cases, following negative results from the IgE-specific in vitro test, the delayed hypersensitivity of the animal to Der HMW-map protein can be tested using an skin test of the present invention.
The present invention also includes kits to detect antibodies that bind specifically to Der HMW-map protein based on each of the disclosed detection methods. One embodiment is a kit to detect Der HMW-map protein-specific antibodies comprising Der HMW-map protein and a means for detecting an IgE
and/or an IgG. Suitable means of detection include compounds disclosed herein that bind to either the Der HMW-map protein or to an IgE and/or an IgG. A preferred kit of the present invention further comprises a detection means including an antibody capable of selectively binding to an IgE or IgG disclosed herein and/or a compound capable of binding to a detectable marker conjugated to a Der HMW-map protein (e.g., avidin, streptavidin and ImmunoPureO NeutrAvidin when the detectable marker is biotin).
Another preferred kit of the present invention is an allergen kit comprising Der HMW-map protein and an allergen commonly detected in the same environment as mite allergen. Suitable and preferred mite-related allergens for use with the present kit include those mite-related allergens disclosed herein.
A preferred kit of the present invention includes those in which Der HMW-map protein is immobilized on a substrate. If a kit comprises Der HMW-map protein and another allergen, the kit can comprise one or more compositions, each composition comprising one allergen. As such, each allergen can be tested separately.
A kit can also contain two or more diagnostic reagents for IgE or IgG, or other compounds as disclosed herein. Particularly preferred are kits used in a lateral flow assay format. It is within the scope of the present invention that a lateral flow assay kit can include one or more lateral flow assay apparatuses. Multiple lateral flow apparatuses can be attached to each other at one end of each apparatus, thereby creating a fan-like structure.
Another aspect of the present invention includes treating animals susceptible to or having mite allergy, with a Der HMW-map protein formulation of the present invention. According to the present invention, the term treatment can refer to the regulation of a hypersensitive response by an animal to mite allergens.
Regulation can include, for example, immunomodulation of cells involved in the animal's hypersensitive response. Immunomodulation can include modulating the activity of molecules typically involved in an immune response (e.g., antibodies, antigens, major histocompatibility molecules (MHC) and molecules co-reactive with MHC
molecules). In particular, immunomodulation refers to modulation of antigen:antibody interactions resulting in inflammatory responses, immunosuppression, and immunotolerization of cells involved in a hypersensitive response.
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.
One embodiment of the present invention is a therapeutic composition that includes desensitizing compounds capable of inhibiting an immune response to Def HMW-map protein of the present invention. Such desensitizing compounds include blocking compounds, toleragens and/or suppressor compounds. Blocking compounds comprise compounds capable of modulating antigen:antibody interactions that can result in inflammatory responses, toleragens are compounds capable of immunotolerizing an animal, and suppressor compounds are capable of immunosuppressing an animal. A desensitizing compound of the present invention can be soluble or membrane-bound. Membrane-bound desensitizing compounds can be associated with biomembranes, including cells, liposomes, planar membranes or micelles. A soluble desensitizing compound of the present invention is useful for: (1) inhibiting a Type I hypersensitivity reaction by blocking IgE:antigen mediated de-granulation of mast cells; (2) inhibiting a Type III hypersensitivity reaction by blocking IgG:antigen complex formation leading to complement destruction of cells;
and (3) inhibiting a Type IV hypersensitivity reaction by blocking T helper cell stimulation of cytokine secretion by macrophages. 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. Examples of 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 desensitizing compound of the present invention can also be covalently linked to a ligand molecule capable of targeting the desensitizing compound to a specific cell involved in a hypersensitive response to Def~ HMW-map protein.
Appropriate ligands with which to link a desensitizing compound include, for example, at least a portion of an immunoglobulin molecule, cytokines, lectins, heterologous allergens, CD8 molecules or major histocompatibility molecules (e.g., MHC class I or MHC class II molecules). Preferred portions of immunoglobulin molecules to link to a desensitizing compound include variable regions capable of binding to immune cell specific surface molecules and constant regions capable of binding to Fc receptors on immune cells, in particular IgE constant regions.
Preferred CD8 molecules include at least the extracellular functional domains of the a chain of CDB. An immune cell refers to a cell involved in an immune response, in particular, cells having MHC class I or MHC class II molecules. Preferred immune cells include antigen presenting cells, T cells and B cells.

In one embodiment, a therapeutic composition of the present invention , includes Der HMW-map protein of the present invention, a mimetope or mutein thereof, or a Der HMW-map nucleic acid molecule of the present invention.
Suitable therapeutic compositions of the present invention for treating mite allergy include Der HMW-map protein, a mimetope or mutein thereof, or a Der HMW-map nucleic acid molecule of the present invention. Preferred therapeutic compositions include:
an isolated mite allergenic protein encoded a nucleic acid molecule that hybridizes under stringent hybridization conditions with the, complement of a nucleic acid molecule that encodes an amino acid sequence selected from the group consisting of SEQ ID
NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID
N0:7, SEQ ID N0:8, SEQ ID N0:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID N0:13, SEQ ID N0:15, SEQ ID N0:18, SEQ ID N0:21, SEQ ID N0:23, SEQ ID N0:24, SEQ ID NO:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID NO:33, SEQ ID NO:35, SEQ ID N0:38, SEQ ID N0:41, and SEQ m N0:44;
a mimetope of the mite allergenic protein; a mutein of the mite allergenic protein; and 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 1X SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid sequence selected from the group consisting of SEQ ID N0:14, SEQ ID
N0:16, SEQ ID NO:17, SEQ ID N0:19, SEQ ID N0:20, SEQ ID N0:22, SEQ ID N0:34, SEQ ID N0:36, SEQ ID N0:37, SEQ II7 N0:39, SEQ ID N0:40, SEQ ID N0:42, SEQ ID N0:43, SEQ m N0:45, and a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID N0:33 and a complement thereof; and a nucleic acid molecule comprising a fragment of any of said nucleic acid molecules comprising at least about 150 nucleotides. A preferred Der HMW-map mutein comprises at least a portion of Def~ 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).

In another embodiment, 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 of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent. A naked nucleic acid of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a bicistronic recombinant molecule having, for example one or more internal ribosome entry sites. Preferred naked nucleic acid molecules include at least a portion of a viral genome (i.e., a viral vector). Preferred viral vectors include those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses, with those based on alphaviruses (such as Sindbis or Semliki virus), species-specific heipesviruses and species-specific poxviruses being particularly preferred. Any suitable transcription control sequence can be used, including those disclosed as suitable for protein production. Particularly preferred transcription control sequence include cytomegalovirus intermediate early (preferably in conjunction with Intron-A), Rous Sarcoma Virus long terminal repeat, and tissue-specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of "strong"
poly(A) sequences are also preferred.
Naked nucleic acid molecules of the present invention can be administered by a variety of methods. Suitable delivery methods include, for example, intramuscular injection, subcutaneous injection, intradermal injection, intradermal scarification, particle bombardment, oral application, and nasal application, with intramuscular injection, intradermal injection, intradermal scarification and particle bombardment being preferred, and intramuscular injection being even more preferred. A
preferred single dose of a naked DNA molecule ranges from about 1 nanogram (ng) to about milligram (mg), depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Examples of administration methods are disclosed, for example, in U.S. Patent No. 5,204,253, by Bruner, et al., issued April 20, 1993, PCT Publication No. WO 95/19799, published July 27, 1995, by McCabe, and PCT Publication No. WO 95/05853, published March 2, 1995, by Carson, et al.
Naked DNA molecules of the present invention can be contained in an aqueous excipient (e.g., phosphate buffered saline) and/or with a carrier (e.g., lipid-based vehicles), or it can be bound to microparticles (e.g., gold particles).
A recombinant virus of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packaging deficient and/or encodes an attenuated virus. A number of recombinant viruses can be a used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses and retroviruses. Preferred recombinant viruses are those based on alphaviruses (such as Sindbis virus), raccoon poxviruses, species-specific herpesvimses and species-specific poxviruses. An example of methods to produce and use alphavirus recombinant virus is disclosed in PCT
Publication No. WO 94/17813, by Xiong et al., published August 18, 1994.
When administered to an animal, a recombinant virus of the present invention infects cells within the recipient animal and directs the production of a protein or RNA
nucleic acid molecule that is capable of reducing Der HMW-map protein-mediated biological responses in the animal. For example, a recombinant virus comprising a Des 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 104 to about 1 x 10' 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 recombinant cells for this embodiment include Salfnonella, E. coli, Listeria, Mycobacterium, S. frugiperda, yeast, (including Saccharomyces cerevisiae ahd Pichia pastoris), BHK, CV-1, myoblast G8, COS (e.g., COS-7), Vero, MDCK and CRFK
recombinant cells. Recombinant cell vaccines 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 10g to about 1012 cells per kilogram body weight. Administration protocols are similar to those described herein for protein-based vaccines. Recombinant cell vaccines can comprise whole cells, cells stripped of cell walls or cell lysates.
The efficacy of a therapeutic composition of the present invention to desensitize an animal against mite allergy can be tested in a variety of ways including, but not limited to, using in vivo skin test methods disclosed herein, detection of cellular immunity activity in the treated animal, or determine levels of IgE
that bind specifically to a Der HMW-map protein of the present invention. Methods to determine cellular immunity activity and IgE levels in an animal are known to those of skill in the art. In one embodiment, therapeutic compositions can be tested in animal models such as dogs, cats, rabbits and mice, and can also be tested in humans.
Such techniques are known to those skilled in the art.
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 N0:14, SEQ ID N0:16, SEQ ID N0:17, SEQ m N0:19, SEQ
ID N0:20, SEQ ID N0:22, SEQ ID N0:34, SEQ ID N0:36, SEQ ID N0:37, SEQ ID
N0:39, SEQ ID N0:40, SEQ ID N0:42, SEQ ID N0:43, SEQ ID N0:45 and/or a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ m N0:33 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 Sahnoizella, E. coli, Listeria, MycobacteYiu~a, S. frugiper-da, 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 108 to about 1012 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.
One embodiment of the present invention is a method of immunotherapy comprising administering to an animal an effective amount of a therapeutic composition comprising a Der HMW-map protein of the present invention.
Suitable therapeutic compositions and methods of administration are disclosed herein.
According to the present invention, a therapeutic composition and method of the present invention can be used to prevent or alleviate symptoms associated with mite allergen pathogenesis.
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.
Therapeutic compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such 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. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
In one embodiment of the present invention, a therapeutic composition can include an adjuvant. Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (e.g., granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), Flt-3 ligand, erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGIF), transforming growth factor beta, RANTES (regulated upon activation, normal T cell expressed and presumably secreted), macrophage inflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmania elongation initiating factor (LEIF); bacterial components (e.g., endotoxins, in particular superantigens, exotoxins and cell wall components); aluminum-based salts;
calcium-based salts; silica; polynucleotides; toxoids; serum proteins, viral coat proteins; block copolymer adjuvants (e.g., Hunter's TitermaxTM adjuvant (VaxcelTM, Inc.
Norcross, GA), Ribi adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, MT); and saponins and their derivatives (e.g., Quil A (Superfos Biosector A/S, Denmark). 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.
In one embodiment of the present invention, a therapeutic composition can include a carrier. Carriers include compounds that increase the half life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.
One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems.
Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).
A preferred controlled release formulation of the present invention is capable of releasing a therapeutic composition of the present invention into the blood of an animal at a constant rate sufficient to attain therapeutic dose levels of the composition to reduce mite allergy in the animal. As used herein, mite allergy refers to cellular responses that occur when mite allergens contact an animal. For example, IgE
that specifically binds to mite allergen becomes coupled with Fc epsilon receptor, resulting in Fc epsilon receptor-mediated biological response including release of biological mediators, such as histamine, prostaglandins and/or proteases; that can trigger clinical symptoms of allergy. The therapeutic composition is preferably released over a period of time ranging from about 1 to about 12 months. 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. For example, 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 hyper sensitive 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. For example, 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. For example, 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). 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 andlor economic food animals. Particularly preferred animals to protect are felines and canines.
Another aspect of the present invention includes a method for prescribing treatment for animals susceptible to or having hypersensitivity to mite allergens, using a formulation of the present invention. A preferred method for prescribing treatment for mite allergen hypersensitivity, for example, 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 wheat 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 vivo 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. A preferred method to monitor treatment of mite allergen hypersensitivity (which can also be adapted to monitor treatment of other allergies) comprises: (1) intradermally injecting an animal at one site with an effective amount of a formulation containing mite allergen, or a mimetope thereof (suitable and preferred formulations are disclosed herein); (2) intradermally injecting an effective amount of a control solution into the animal at a second site; and (3) determining if the animal is desensitized to mite allergens by measuring and comparing the wheat size resulting from injection of the formulation with the wheat size resulting from injection of the control solution.
An alternative preferred method to monitor treatment of mite allergen hypersensitivity (which can be adapted to monitor treatments of other allergies) 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 a mite allergen or 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; and (3) determining if the animal is desensitized to mite allergens by measuring and comparing the amount of immunocomplex formation in the first and second immunocomplex solutions.
The present invention also includes antibodies capable of selectively binding to mite allergen, or mimetope thereof. Such an antibody is herein referred to as an anti-mite allergen antibody. As used herein, the term "selectively binds to" refers to the ability of such an antibody to preferentially bind to mite allergens and mimetopes thereof. In particular, 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.
Antibodies of the present invention can be either polyclonal or monoclonal antibodies. Antibodies of the present invention include functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protein or mimetope used to obtain the antibodies. Preferred antibodies are raised in response to Der HMW-map proteins, or mimetopes thereof. More preferred Der HMW-map protein against which to raise an antibody includes at least a portion of a protein having the amino acid sequence SEQ ID NO:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, SEQ ID N0:8, SEQ ID
N0:9, SEQ ID NO:10, SEQ ID NO:l 1, SEQ ID N0:12, SEQ ID N0:13, SEQ ID
N0:15, SEQ ID N0:18, SEQ ID N0:21, SEQ ID N0:23, SEQ ID N0:24, SEQ ID
N0:30, SEQ ID N0:31, SEQ ID N0:32, SEQ ID N0:33, SEQ ID N0:33, SEQ ID
N0:35, SEQ ID N0:38, SEQ ID N0:41, and/or SEQ ID N0:44, or homologs thereof.
Preferably, an antibody of the present invention has a single site binding affinity of from about 103M-1 to about 1012M-1 for a Der HMWWmap protein of the present invention.
A preferred method to produce antibodies of the present invention includes administering to an animal an effective amount of a Der HMW-map protein or mimetope thereof to produce the antibody and recovering the antibodies.
Antibodies raised against defined products 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. For example, such antibodies can be used (a) as vaccines to passively immunize an animal in order to protect the animal from mite allergen hypersensitivity, (b) as positive controls in test kits, and/or (c) as tools to recover desired mite allergens from a mixture of proteins and other contaminants.
The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention.
EXAMPLES
It is to be noted that the Examples include a number of molecular biology, microbiology, immunology and biochemistry techniques considered to be known to those skilled in the art. Disclosure of such techniques can be found, for example, in Sambrook et al., ibid., and related references.
Example 1 This example describes the identification of high molecular weight proteins that bind to IgE from dogs known to be allergic to mite allergens.
About 5.5 grams (g) of frozen wet Dermataplzagoides farazzae (Derv mites (available from Bayer Allergy, Spokane, WA) were homogenized in a ground glass homogenizer, in either about 30 ml of phosphate buffered saline (PBS) or 0.1 M
Tris-HCI, pH ~, each containing complete protease inhibitors (available from Boehringer Mannheim, Indianapolis, IN) to obtain a Der f crude extract. The resulting supernatants were collected and each concentrated in a Centriprep 30 concentrator (available from Amicon, Beverly, MA) by centrifugation at 16,000 x g for about minutes. The concentrated supernatants were applied to separate Sephacryl S-columns (2.7 x 70 cm; available from Pharmacia, Piscataway, NJ) in PBS or 0.1 M
Tris-HCl, pH 8, respectively. The excluded fractions from each column were pooled.
Fractions were dialyzed against 10 mM Tris-HCI, pH 8, when PBS was used. The fractions were applied to separate Q-Sepharose columns (2.5 x 5 cm; available from Pharmacia). The Q-Sepharose column was pre-equilibrated in 10 mM Tris-HCl, pH
8, when the fractions containing 0.1 M Tris-HCI, pH 8 were used. Each column was sequentially eluted with about 45 ml of 10 mM Tris-HCl, pH 8, then 0.1 M Tris-HCI, pH 8, then 0.2 M Tris-HCI, pH 8, then 0.3 M Tris-HCI, pH 8, then 0.4 M Tris-HCI, pH
8 and then 0.5 M Tris-HCI, pH 8. Fractions were collected from each elution step.
Each fraction was analyzed by western blot for the presence of protein that bound to IgE antibodies present in dog sera isolated from dogs known to be allergic to mite allergens (referred to herein as mite allergic dog antisera or mite allergic antisera).
Specifically, proteins contained in the fractions were resolved by 12°70 Tris-glycine SDS-PAGE and then blotted onto nitrocellulose. The blot was incubated with a pool of sera obtained from dogs known to be allergic to mite allergens, diluted 1:20, using standard buffers. The blot was incubated and then washed using standard procedures.
The blot was then incubated with the mouse monoclonal anti-dog IgE antibody (1 mg/ml, 1:1000 dilution). The blot was incubated and then washed using standard procedures. 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-HCI, pH 8 fraction.
The fraction described above that was eluted using 0.3 M Tris-HCI, 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 canon exchange column (available from PoIyLC, 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. ' Fractions were collected from the elution procedure and assayed for the presence of high molecular weight proteins using the mite allergic antisera and western blot protocol described above. Fractions containing the high molecular weight pr oteins were pooled. Trifluoroacetic acid (TFA) was added to a concentration of about 0.05%.
The solution was applied to a TSK-Gel TMS-250 Cl reverse phase column (available from TosoHaas, Montgomeryville, PA) that had been pre-equilibrated in 80%
solvent A and 20% solvent B. Solvent A was composed of about 0.05% TFA in water and solvent B was composed of about 0.05% TFA in 90% acetonitrile in water. The column was eluted with about 5 ml of 20% solvent B and then with 36 ml of a linear gradient of about 20% to about 70% solvent B at 0.6 ml/min. The proteins eluted from the column were resolved by 12% Tris-Glycine PAGE. The gel, was stained with Comassie blue. The stained gel is shown in Fig. 1. Lane 1 contains Mark-12 protein molecular weight markers (available from Novex, San Diego, CA), lane 2 contains the protein eluted from the reverse phase column, and lane 3 contains SeeBlueT"~
protein molecular weight markers (available from Novex). Two major proteins were identified in the eluant. The molecular weights of the proteins were determined using a BioRadTM Multi-AnalystTM/PC Image System (available from BioRad Corp.). The higher molecular weight protein in lane 2 of Fig. 1 was determined to be about kD, referred to herein as mite allergen protein A (mapA). The lower molecular weight protein in lane 2 of Fig. 1 was determined to be about 98 kD, referred to herein as mite allergen protein B (mapB). The purity of the combined proteins was greater than 85%
purity, i.e., less than 15% impurities. This purified eluant is referred to herein as the D. fariraae high molecular weight map (HMW-map) composition.
Example 2 This example describes N-terminal sequencing of proteins in the D. fariaae HMW-map composition.
Proteins contained in the 0.3 M Tris-HCI, pH 8 fraction obtained as described above in Example 1 were resolved by SDS-PAGE using a 12% Tris-glycine polyacrylamide-SDS gel, followed by coomasie staining. The proteins were blotted onto PVDF, stained with Coomasie R-250 and destained using standard procedures.
The proteins corresponding to the about 98 kD and about 109 kD bands were excised and subjected separately to N-terminal amino acid sequencing using techniques known to those skilled in the art. A partial N-terminal amino acid sequence of about amino acids was deduced for both proteins and the sequences were determined to be identical. The N-terminal amino acid sequence is represented herein as SEQ m NO:1, having the amino acid sequence: Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met.
The proteins in the D. fariTaae HMW-map composition were also submitted to proteolytic cleavage in order to obtain internal amino acid sequence data.
Specifically, the D. farihae HMW-map composition was cleaved with Endoproteinase Asp-N
(available from Boehringer Mannheim Biochemica, Indianapolis, IN) using methods standard in the art. The digested protein was then resolved by HPLC using the method described by Stone et al., Enzymatic Digestion of Proteins and HPLC Peptide Isolation, in A Practical Guide to Protein and Peptide Purification for Microsequencing, PT Matsudaira ed., Academic Press, San Diego, CA. Twelve proteolytic fragments were isolated, that are referred to herein as map(1), map(2), map(3), map(4), map(5), map(6), map(7), map(8), map(9), map(10), map(11) and map(12).
The N-terminal partial amino acid sequence of map(1) was determined to be Asp Tyr Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ala Pro Leu Tyr Lys Arg Pro, also denoted SEQ ID N0:2. The N-terminal partial amino acid sequence of map(2) was determined to be Asp Ile Pro His Pro Thr Asn Ile His Lys Tyr Leu Val Cys Glu Ser Val Asn Gly Gly, also denoted SEQ 117 N0:3. The N-terminal partial amino acid sequence of map(3) was determined to be Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Val Gly Glu Glu Gly Val Leu Ser, also denoted SEQ ID N0:4. The N-terminal partial amino acid sequence of map(4) was determined to be Asp Glu Lys Asn Ser Phe Glu Cys Ile Leu Gly Pro, also denoted SEQ ID N0:5. The N-terminal partial amino acid sequence of map(5) was determined to be Asp Ala Phe Glu Pro His Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly Lys, also denoted SEQ ID N0:6. The N-terminal partial amino acid sequence of map(6) was determined to be Asp Lys Gln Asn Tyr Leu Ala Leu Val Arg Glu Leu Lys, also denoted SEQ ID N0:7. The N-terminal partial amino acid sequence of map(7) was determined to be Asp Met Ala Gln Asn Tyr Lys Tyr Arg Gln Gln Phe Ile Gln Ser Val Leu Asn Asn Gly Ala Thr Arg Gln, also denoted SEQ ID N0:8. 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 N0:9, in which Xaa represents any amino acid. The N-terminal partial amino acid sequence of map(9) was determined to be Asp Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Xaa Ser Ile Glu, also denoted SEQ ll~ NO:10, in which Xaa represents any amino acid. The N-terminal partial amino acid sequence of map(10) was determined to be Asp Ile Pro His Pro Thr Asn Ile His Lys Tyr Leu Val Cys Glu Ser Val Asn Gly, also denoted SEQ
ID
N0:11. The N-terminal partial amino acid sequence of map(11) was determined to be Asp Tyr Ala Lys Asn Pro Lys Arg Ile Val Cys Ile Val Gly Thr Glu Gly Val Leu Ser, also denoted SEQ ID N0:12. The N-terminal partial amino acid sequence of map(12) was determined to be Asp Pro Ala Lys Gly Met Ser Pro Pro Gly He Ile Val Gly Glu Glu Gly Val Leu Ser, also denoted SEQ ID N0:13. Since the amino acid sequences for map(1), map(2), map(3), map(4), map(5), map(6), map(7), map(8), map(9), map(10), map(11), map(12), and map(13) were generated from a mixture of mapA
and mapB proteins, these sequences do not necessarily represent partial sequences of a single protein.
Example 3 This example describes the purification of a 70-kD protein that binds to IgE
from dogs known to be allergic to mite allergens.
The fraction described above in Example 1 that was eluted using 0.2 M
Tris-HCI, pH 8 was concentrated in a Centriprep 30 concentrator and then diluted in 20 mM Na-Ac, pH 5.6. The diluted protein was then applied to a PolyCat A HPLC
cation exchange column. 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 NaCI in the 20 mM Na-Ac, pH 5.6 buffer at a flow rate of about 1 ml/min. Fractions were collected from the elution procedure and assayed for the presence of 70-kD protein using the mite allergic antisera and western blot protocol described above. Fractions containing the 70-kD protein were pooled. Trifluoroacetic acid (TFA) was added to a concentration of about 0.05%. The solution was applied to a TSI~-Gel TMS-250 reverse phase column that had been pre-equilibrated in 80% solvent A and 20%

_72_ solvent B. Solvent A was composed of about 0.05% TFA in water and solvent B
was composed of about 0.05% TFA in 90%o acetonitrile in water. The column was eluted with about 3 ml of 20% solvent B and then with 36 ml of a linear gradient of about 20% to about 70% solvent B at 0.6 ml/min. An about 70-kD protein of >90%
purity was obtained. The about 70-kD protein is referred to herein as mapC.
N-terminal sequence of a region on an SDS-PAGE corresponding to the 70 kD
protein (mapC) was obtained as described in Example 2. An N-terminal amino acid sequence of about 21 amino acids was deduced with an 80% confidence level, and is represented herein as SEQ ID N0:33, having the following amino acid sequence:
Gln Ser Arg Asp Arg Asn Asp Lys Pro Tyr Xaa Ile Val Lys Lys Lys Lys Lys Ala Leu Asp.
Example 4 This example describes the binding of the D. faz~iyzae 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 II microtiter plate were coated with about 100 nanograms per well (nglwell) of a D. fat ireae HMW-map composition isolated according to the method described above in Example l, diluted in CBC buffer.
The plate was incubated overnight at 4°C. Following incubation, the D.
farin.ae HMW-map composition-containing solution was removed from the plate, and the plate was blotted dry. The plate was then blocked using about 200 ,ul/well of 4.0% fetal calf serum contained in phosphate buffered saline (PBS) having 0.05% Tween-20 (PBSTFCS) for about 1 hour at room temperature. The plate was then washed four times with 0.05% Tween-20 in PBS (PBST) using an automatic washer (available from Dynatech, Chantilly, VA). About 100 ,ul/well of a 1:10 dilution in PBSTFCS of serum samples isolated from different dogs known to be sensitive to mite allergens in intradermal skin tests were added to the plate. 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. About 100 ,ul/well of a 1:4000 dilution of 40 ,ug/ml biotinylated human FcER

alpha chain protein (produced as described in Frank et al., WO 98/23964, published November 24, 1997) contained in PBSTFCS was added. The plate was incubated for about 1 hour at room temperature and then washed four times with PBST. About ,u! of about 0.25 ,ug/ml streptavidin conjugated to horseradish peroxidase (available from Kirkegaard and Perry Laboratories (KPL), Gaithersburg, MD; diluted in PBST) was added to each well that received experimental or control samples. The plates were then incubated for about 1 hour at room temperature and washed four times with PBST. About 100 ,u! of TMB substrate (available from KPL), that had been pre-warmed to room temperature, was added to each well. The plate was then incubated for about 10 minutes at room temperature and then about 100 ,ul/well of Stop Solution (available from KPL) was added. Optical densities (0.D.) of wells were read on a Spectramax Microtiter Plate (available from Molecular Devices Inc.) reader at 450 nm within 10 minutes of adding the stop solution.
The O.D. readings obtained using the negative control sample and the background wells were 0 O.D. Sera from 5 of 26 mite allergen sensitive dogs generated O.D. readings between about 2,000 O.D. and about 3,200 O.D. Sera from 3 other mite allergen sensitive dogs generated O.D. readings between about 1,000 O.D.
and 2,000 O.D. Sera from 3 other mite allergen sensitive dogs generated O.D.
readings between about 500 O.D. and 1,000 O.D. Sera from 7 other mite allergen sensitive dogs generated O.D. readings between about 200 O.D. and 500 O.D.
Sera from 6 other mite allergen sensitive dogs generated O.D. readings less than 50 O.D.
Thus, the results indicate that sera from dogs known to be sensitive to mite allergens contain IgE antibodies that bind specifically to the mapA and mapB proteins of the present invention.
Example 5 This example describes the binding of the 70-kD D. farihae protein to canine IgE in dog sera isolated from dogs known to be allergic to mite allergens.
Multiple wells of an Immulon II microtiter plate were coated with about 100 nglwell of 70-kD D. fariTZae protein (referred to herein as mapC) isolated according to the method described above in Examples 1 and 3, diluted in CBC buffer. The plate was incubated overnight at 4°C. The plate was blocked and washed using the method described in Example 4. About 100 ,ul/well of a 1:10 dilution in PBSTFCS of serum samples isolated from different dogs known to be sensitive to mite allergens in intradermal skin tests were added to the plate. Negative control samples were also added to the plate comprising SPF serum samples (serum from dogs maintained in a barrier facility and therefore never exposed to mite allergens). 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 PEST. Biotinylated human FcER alpha chain protein was then added and the presence of IgE bound to the plate was detected using the methods described in Example 4.
The O.D. readings obtained using the negative control sample and the background wells were 0 O.D. Sera from 3 of 26 mite allergen sensitive dogs generated O.D. readings between about 1,500 O.D. and about 2,700 O.D. Sera from 5 other mite allergen sensitive dogs generated O.D. readings between about 800 and about 1,500 O.D. Sera from 4 other mite allergen sensitive dogs generated O.D.
readings between about 500 O.D. and about 800 O.D. Sera from 6 other mite allergen sensitive dogs generated O.D. readings between about 200 O.D. and 500 O.D.
Sera from 8 other mite allergen sensitive dogs generated O.D. readings less than 50 O.D.
Thus, the results indicate that sera from dogs known to be sensitive to mite allergens contain IgE antibodies that bind specifically to the mapC protein of the present invention.
Example 6 This example describes the binding of mapA, mapB or mapC proteins to feline IgE in eat sera isolated from cats shown by ira vitro testing to be hypersensitive to mite allergens.
Multiple wells of an Tmmulon II microtiter plate were coated with about 100 ng/well of a D. farircae HMW-map composition (isolated according to the method described above in Example 1) and 70-kD D. farifaae protein (isolated according to the method described above in Example 3). Other wells of the plate were coated with 400 ng/well of whole Derfnatophagoides pteronyssius extract (available from Greer Laboratories, Inc., Lenoir, NC; concentrated 8-fold prior to use) or whole D.
farifZae extract (available from Miles, Inc., Elkhart, IN). All samples were diluted in CBC

buffer. The plates were incubated overnight at 4°C. The plates were blocked and washed using the method described in Example 4. About 100 ,ul/well of a 1:10 dilution in PBSTFCS of serum samples isolated from different cats known to be sensitive to mite allergens in i~z vitro allergen testing were added to the plate. Sera from seven control cats (#15, #16, #17, #18, #19, #20, and #21), shown not to be sensitive by ire vitro test to dust mite allergens, were also tested. Some wells did not receive cat 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. Biotinylated human FcER alpha chain protein was then added and the presence of IgE bound to the plate was detected using the methods described in Example 4.
The results are shown below in Table 1. All values represent O.D. values times 1,000. HDM refers to cats that are sensitive to house dust mite allergens (by serological test, i.e. an ELISA to whole D. fariaae extract).
Table 1.
Cat HDM Whole Der Whole Der mapA and mapBmapC
# p ' f 1 + 54 173 211 400 2 + 437 454 245 352 3 + 96 88 17 36 4 + 35 179 278 758 5 + 123 23 0 0 6 + 2 10 0 0 7 + 84 321 439 445 8 + 125 333 611 599 9 + 2459 2737 1613 507 10 + 17 0 0 0 11 + 146 347 243 586 12 + 31 100 102 223 13 + 56 171 267 292 14 + 121 146 163 185 The results indicate that sera from some of the cats known to be sensitive to mite allergens contain IgE antibodies that bound specifically to the mapA, mapB or mapC
proteins of the present invention. In addition, some sera containing IgE that bound to the mapA, mapB or mapC proteins also contain IgE antibodies that bound to whole D.
pterouyssius extract. The control sera did not contain IgE antibodies that bound to either the mapA, mapB or mapC proteins of the present invention.
Example 7 This example demonstrates the ability of the D. faf-iozae HMW-map composition to induce a hypersensitive response in dogs.
To determine whether the D. fariT2ae HMW-map composition described in Example 1 was capable of inducing an allergic response in animals susceptible to dust mite allergic responses, skin tests were performed on dogs that actively demonstrate clinical signs for dust mite allergy (referred to herein as atopic dogs).
Normal dogs include dogs that do not show symptoms of mite allergy but may be susceptible to a mite allergic response. Each dog (i.e., 4 normal and 4 atopic dogs) was shaved in the lateral thorax/abdominal area and intradermally injected in different sites in that area with an about 1:50,000 dilution of D. fariuae crude extract isolated by the method described in Example l, with about 2 ,ug of the purified D. fari~za.e HMW-map composition and/or with control solutions, i.e., saline, as a negative control, and a 1:1000 dilution of histamine as a positive control. All four normal dogs and all 4 atopic dogs received D. farihae whole extract. Three of the normal dogs and 2 of the atopic dogs received the D. faf~is2ae HMW-map composition. All 8 of the dogs received both the negative and positive control samples. The total volume per injection was 50 microliters (,u1), with the compositions and controls being diluted in saline. The injections were administered as single injections.
All injection sites were objectively measured in millimeters (mm) at 15 minutes and scored either (+) or (-) when compared with the control samples.
The subjective scoring was performed by Andrew Hillier, D.V.M., at Ohio State University, Columbus, OH. The results are shown in Table 2:

_77_ Table 2.
NdrmalNormal:.Normal: NormalAtopicAtopic AtopieAtopic Dog'1.I)og.2 Dog Dog Dog Dog Dog Dog ' 3. 4 1 2 3 4 Whole Extract+ + + - + + - -HMW map + + - n/a + - n/a n/a Neg. Control- - - - - - - -Histamine + + + + + + + +

n/a = not applicable The results indicate that the D. faritZae HMW-map composition was capable of inducing an immediate hypersensitive response in dogs including atopic dogs.
Thus, the HMW-map composition is sufficiently allergenic to induce a hypersensitive response in dogs including atopic dogs.
Table 3 describes the results of the following experiment. IgE to the HMW-map composition was measured in the serum of three groups of dogs: D. farin.ae allergic (HDM-AD), atopic (to other allergens) but not HDM allergic (AD), and naive dogs using ELISA. These dogs were also tested by intradermal skin test to D.
farii2ae whole extract and to the HMW-map composition.
Table 3. Skin test and ELISA data for D. farioae whole extract and for HMW-map composition in D. farihae-allergic, atopic but not HDM-allergic, and naive dogs.
Dog Clinical Df IDST Df ELXSA:~y~!-mapHMW-map status 1:50;000 IDST'1u ELTSA
:

1 HDM-AD + 1968 + 2876 2 HDM-AD + 407 - 954 3 HDM-AD + 3921 + 3465 4 HDM-AD + 153 + 198 5 HDM-AD + 1712 + 997 6 HDM-AD + 1833 + 2006 7 HDM-AD + 4200 + 4200 8 HDM-AD + 2851 + 3559 9 HDM-AD + 122 + 209 10 HDM-AD + 1627 + 566 11 HDM-AD + 1185 + 1307 12 HDM-AD + 308 + 101 13 HDM-AD + 341 + 433 _78_ Dog Clinical Df IDST Df ELISAHMWmap. HMW-map status 1:50,000 IDST 1u ELISA
v 17 Normal - 24 - 40 18 Normal - 53 ND 369 19 Normal - 37 - 21 20 SPF bea ND 0 ND 0 1e 21~ SPF beagleND I 6 ND 1 I

All dogs that were positive by ELISA for whole D. faf°ihae extract were also positive for the HMW-map composition allergen. Of the eight dogs that were ELISA
negative for whole D. farireae extract, 7 of 8 were also negative for the HMW-map composition.
Example 8 This example describes the isolation of nucleic acid molecules encoding a Der HMW-map composition of the present invention.
Der HMW-map composition nucleic acid molecules were identified and isolated as follows.
A. Preparation of a Dermatophagoides farinae cDNA Library.
A Dermatophagoides fariizae cDNA library was prepared as follows. Total RNA was extracted from about 2 grams of flash frozen and pulverized house dust mites, using an acid-guanidinium-phenol-chloroform method similar to that described by Chomzynski et al., 1987, A~eal. Biochen2. 162,156-159. Poly A+ selected RNA
was separated from the total RNA preparation by oligo-dT cellulose chromatography using the mRNA Purification Kit ( available from Pharmacia Biotech, Newark, NJ), according to the method recommended by the manufacturer. A cDNA library was constructed in lambda-Uni-ZAPTM XR vector (available from Stratagene), using Stratagene's ZAP-cDNA Synthesis Kit protocol. Approximately 5 ~,g of Poly A~
RNA
was used to produce the Dermatoplzadoides farin.ae cDNA library.
B. Preparation of PCR primers.
Further N-terminal amino acid sequence analysis was performed according to the methods described above in Example 2. A partial N-terminal amino acid sequence of 34 amino acids was deduced and is represented by SEQ ID N0:24, having the amino acid sequence: Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met Met Ile Val Xaa Tyr Tyr Gly Gly Ser Ser Gly Tyr Gln Ser Xaa Lys Arg Xaa Xaa Thr (wherein "Xaa" represents any amino acid residue). The amino acid sequences of SEQ
ll~ N0:4 (described above in Example 2) and SEQ ID N0:24 were used to design synthetic oligonucleotide primers. Sense primer Derfl derived from SEQ m N0:24, having the nucleotide sequence 5' AAA CGT GAT CAT AAY. GAT TAY TCN AAR
AAY C 3' (wherein Y represents C or T, R represents A or G, and N represents A, C, T or G), designated SEQ ID NO: 25 or sense primer Derf2, derived from SEQ ID
N0:24, having the nucleotide sequence 5' AAA CGT GAT CAT AAY GAT TAY
AGY AAR AAY C 3', designated SEQ ID N0:26, were used in combination with antisense primer Derf3 deriveel from SEQ ID N0:4, having the nucleotide sequence 5' CCT TCT TCA CCN ACR ATC AAN CC 3', denoted SEQ 117 N0:27, or antisense primer Derf4 derived from SEQ ID NO:4, having the nucleotide sequence 5' CCT
TCT TCA CCN ACR ATG AAN CC 3', denoted SEQ ID N0:28.
The foregoing primers were then used to screen the Der f cDNA library using standard polymerase chain reaction amplification (PCR) techniques. All attempts to identify a cDNA that hybridized to the primers failed.
C. Immunoscreening the D. farihae cDNA library using anti-Der HMW-mapcomposition antibodies.
Since attempts to isolate a cDNA clone using PCR methods failed, the inventors screened the D. farihae cDNA library using an antiserum produced as follows. Protein isolated according to the method described above in Example 1 was used as a source of antigen to generate rabbit polyclonal antibodies, referred to herein as anti-Der HMW-map composition antibodies. The preparation of rabbit polyclonal antibodies was carried out using standard techniques.
About 7.5 ml of Escherichia coli (XL1 Blue, O.D.boo=0.5) was incubated with 3.0 x 104 pfu of phage from a Dermatophagoides farircae ZAP-cDNA library ( 1.8 x 109pfu/ml), at 37°C for 15 min and plated in 30 ml Luria-Bertani (LB) medium agar plates (150 mm). The plates were incubated at 37°C over night. Each plate was then overlaid with an IPTG (10 mM) treated nitrocellulose filter for about 4 hours at 37°C.
The filters were then removed and washed with Tris buffered saline (pH 7.5) containing 0.1% Tween (TBST), for 5 minutes. The filters were blocked with a solution of 1% dried pwder milk, 1% BSA, 2% goat serum and 0.15% gelatin, prepared in TBST, for 2 hours at room temperature. Filters were then incubated with the anti-Der HMW-map composition antibodies at a dilution of 1:1000, contained in the above blocking solution at 4°C, overnight. The mixture was then incubated with a donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase (available from Jackson ImmunoResearch, West Grove, PN) for 2 hours at room temperature. All of the filters were washed with blocking solution contained in TBST (3 x 15 min/wash) between each incubation. All of the filters were then treated to a final wash in Tris buffered saline (pH 7.5) for 5 minutes at room temperature. Immunocomplexed plaques were identified by immersing the filters into the developing solution (TMB
Peroxidase Substrate/TMB Peroxidase Solution/TMB Membrane Enhancer from I~irkegaard & Perry Laboratories) at 1/1/0.1 volume ratio to produce a color reaction.
One hundred and twenty three plaques were identified and 50 plaques were further plaque purified two more times under the same immunoscreening condition as described above.
D. PCR screening of purified phage plugs The phage plugs identified in the foregoing immunoscreening study were then further analyzed by PCR amplification using the primers described above in section 8B. DNA from the 50 plaques was amplified using a mixture of the 4 primers identified by SEQ ll~ NO: 25, SEQ ID N0:26, SEQ ID N0:27 and SEQ ID N0:28.
PCR amplification was conducted using standard techniques. One resulting PCR
amplification product comprised a fragment of about 700 nucleotides. The PCR
product was cloned into the InVitrogen, Corp., TAT"' cloning vector (procedures provided by InVitrogen, Corp.) and subjected to DNA sequence analysis using standard techniques. The phagemid from the purified phage that were determined to contain sequences encoded in the 700-by PCR product were rescued and subjected to DNA sequence analysis using standard techniques.
A clone was isolated that included about a 1752-nucleotide insert, referred to herein as nDerf981~sz. Nucleic acid sequence was obtained using standard techniques from nDerf981~sz, to yield a Derrnatophagoides farihae nucleic acid molecule named nDerf981~s~ composed of a coding strand having nucleic acid sequence SEQ ID
N0:14 and a complementary strand having a nucleic acid sequence SEQ ID N0:16.
Translation of SEQ ID N0:14 suggests that nucleic acid molecule nDerf981~sz encodes a full-length flea protein of about 555 amino acids, referred to herein as PDerf98sss~
having amino acid sequence SEQ ID N0:15, assuming an open reading frame in which the first codon spans from nucleotide 1 through nucleotide 3 of SEQ ID
N0:14 and a stop codon spanning from nucleotide 1666 through nucleotide 1668 of SEQ
ID
N0:14. The amino acid sequence of PDerf98sss is encoded by the nucleic acid molecule'nDerf98166s, having a coding strand with the nucleic acid sequence SEQ ID
NO:17 and a complementary strand with the nucleic acid sequence SEQ WN0:19.
PDen98sss , also represented by SEQ ID N0:18, has an estimated molecular weight of about 63.2 kD and an estimated pI of about 5.33. Analysis of SEQ ID N0:15 suggests the presence of a signal peptide spanning from about amino acid 1 through about amino acid 19. The proposed mature protein, denoted herein as PDerfs36, contains about 536 amino acids, the sequence of which is represented herein as SEQ ID
N0:21, and is encoded by a nucleic acid molecule referred to herein as nDerf981~os~
represented by SEQ ID N0:20, the coding strand, and SEQ ID N0:22, the complementary strand. The amino acid sequence of flea PDerf98s36 (i.e. SEQ ID
N0:21) predicts that PDerf98s3s has an estimated molecular weight of 61.2 kD, and an estimated pI of about 5.26.
Comparison of amino acid sequence SEQ ID N0:15 with amino acid sequences reported in GenBank indicates that SEQ ID N0:15 showed the most homology, i.e., about 42% identity, with a chitinase protein fromAfzopl~eles gambiae (GenBank accession number 2654602). Comparison of nucleic acid sequence SEQ ID
N0:17 with nucleic acid sequences reported in GenBank indicates that SEQ ID
N0:17 showed the most homology, i.e., about 58% identity between SEQ ID N0:17 and Chelo~us ,rp. venom chitinase mRNA (GenBank accession number U10422).
Example 9 This example describes the purification of a 60-kD protein that binds to IgE
from dogs known to be allergic to mite allergens and partial amino acid sequences derived from this 60-kD protein.

A. Purification of a 60 kD protein D. farihae extract was prepared and fractionated on a Sephacryl S-100 column according to the methods described above in Example 1. Fractions were collected from the Sephacryl S-100 column after the excluded peak (fractions 29 through 35) and were pooled. The pooled fractions were then diluted 1:1 with 10 mM Tris-HCI, pH 8, and applied to a Q-sepharose column and fractions obtained using the methods described above in Example 1. The fraction that eluted in 0.4 M Tris-HCl was concentrated and further purified through a TMS 250 reverse phase HPLC column using the methods described above in Example 1. The proteins in the fractions were resolved by 14°lo Tris-glycine SDS-PAGE using similar methods described for resolution of proteins on the 12% gel in Example 1. The stained gel is shown in Fig.
2. A protein was identified having a molecular weight of about 60 kD (Fig. 2, lane 4) of about 90°Io purity that eluted at about 50% B (.05%TA in 90%
acetonitrile). The molecular weight of the denoted 60-kd protein was estimated to be 56.11 kd using the BioRad Multi-Analyst/PC Version 1.1 program and Mark-12 protein molecular weight markers. The about 60-kd protein is referred to herein as mapD protein.
B. Partial N-terminal and internal sequence obtained from the 60-kd protein The eluted protein from Part A, above, was blotted onto PVDF, which was stained with Coomassie R-250 and destained using standard procedures. The protein corresponding to the about 60-kd band was excised and subjected to N-terminal amino acid sequencing using techniques known to those skilled in the art. A partial N-terminal amino acid sequence of about 25 amino acids was deduced for the protein and the amino acid sequence, represented herein as SEQ TD N0:23, was determined to be: Xaa Leu Glu Pro Lys Thr Val Cys Tyr Tyr Glu Ser Trp Val His His Arg Gln G1y Glu Gly Lys Met Asp Pro (wherein Xaa refers to any amino acid).
The protein corresponding to the 60 kd region was also submitted to proteolytic cleavage in order to obtain internal amino acid sequence data.
Digestion of the 60-kd protein and reverse-phase chromatography were carried out as described in Example 1. Four proteolytic fragments were isolated and sequenced, and are referred to herein as map(13), map(14), map(15), and map(16).
The N-terminal partial amino acid sequence of map(13) was determined to be Gln Tyr Gly Val Thr Gln Ala Val Val Thr Gln ProAla, also denoted SEQ m N0:29.

The N-terminal partial amino acid sequence of map(14) was determined to be Asp Glu Leu Leu Met Lys Ser Gly Pro Gly Pro, also denoted SEQ ID N0:30. The N-terminal partial amino acid sequence of map(15) was determined to be Asp Met Glu His Phe Thr Gln His Lys Gly Asn Ala Lys Ala Met Ile Ala Val Gly Gly Ser Thr Met Ser, also denoted SEQ ID N0:31. The N-terminal partial amino acid sequence of map(16) was determined to be Asp Ala Asn Glu Glu Ala Arg Ser Gln Leu Pro Glu Thr Ala Met Val Leu Ile Lys Ser Gln, denoted SEQ ID N0:32.
Example 10.
This example describes the isolation and sequencing of nucleic acid molecules encoding a portion of the D. farihae 60 kD (mapD) allergen.
A D. farihae library was prepared as described previously in Example 8. A
degenerate synthetic oligonucleotide primer was designed from the N-terminal amino acid sequence deduced for D. farinae 60 kD-protein (SEQ ID N0:23): Primer l, a sense primer corresponding to amino acid residues from about 3 through about 11 of SEQ ID N0:23 has the sequence 5' GAACCAAAA CHGTNTGYTA YTAYG 3', also known as SEQ ID N0:46, where H represents A or C or T, N represents A or C or G
or T, and Y represents C or T. PCR amplification of fragments from the D.
farinae library was conducted using standard techniques. A PCR amplification product was generated using a combination of SEQ ID N0:46 (primer 1) and the M13 forward universal primer 5'GTAAAACGACG GCCAGT 3', denoted SEQ ID N0:47.
A second, nested PCR reaction was carried out on the products of the first PCR
reaction. A synthetic oligonucleotide was synthesized that corresponded to a region spanning from about amino acid residue 1 through amino acid residue 10 of the 60-kD
protein internal amino acid sequence, SEQ ID N0:31. This primer, primer 2, has the nucleic acid sequence 5' GATATGGAAC ATTTYACHCA ACAYAARGG 3', denoted SEQ ID N0:48, where R represents A or G. A PCR amplification product was generated using the combination of primer 2, SEQ ID N0:48, and the T7 standard primer, 5' GTAATACGAC TCACTATAGG GC 3', denoted SEQ ID N0:49. The resultant PCR product Was subjected to DNA sequence analysis using standard techniques.

The PCR product was sequenced and found to contain 510 nucleotides, and is known as nDerf605~o. The nucleotide sequence of the coding strand of nDerf60s~o is represented herein as SEQ m N0:43, and its complement is denoted SEQ ID N0:45.
Translation of SEQ ID N0:43 suggests that nDerf605io encodes a partial D.
faritaae 60-kD protein of about 170 amino acids, referred to herein as PDerf601~o, with an amino acid sequence denoted SEQ ID N0:44, assuming an open reading frame in which the first codon spans from about nucleotide 1 through nucleotide 3 of SEQ ID
N0:43, and the last codon spanning from about nucleotide 508 through about nucleotide 510 of SEQ ID N0:43. PDerf601~o has an estimated molecular weight of 19.2 kD and an estimated pI of about 6.51.
Nucleic acid molecule nDerf605io was used as a probe to isolate a nucleic acid molecule that encodes a protein corresponding to a full-length, or larger partial D.
faYinae 60-kD protein. Using procedures described previously in Example 8, the whole D. farinae library was screened with the nucleic acid SEQ ID N0:43 radiolabeled with 32P using standard techniques. Hybridization was done in 6X
SSC, 5X Denhardt's solution, 0.5% SDS, 100 mglml ssDNA, at 55°C, for about 36 hours.
The filters were washed 3 times, for 30 minutes per wash, at 55°C in 2X
SSC, 0.2%
SDS, followed by a final wash of about 30 minutes in 0.2X SSC, 0.2% SDS.
PCR amplification was carried out on the primary phage plugs. Primer 1, denoted as SEQ ID N0:46, and T7 standard primer, denoted as SEQ ID N0:49, were used as the primers, and a PCR product was generated. Preliminary sequence analysis of this 1.6 kilobase PCR product showed that it represents a nucleic acid sequence that contains the complete sequence encoding the PDerf60 full-length protein.
Comparison of PDerf601~o, the amino acid sequence of SEQ ID N0:44, with amino acid sequences reported in GenBank indicates that PDerf601~o showed the most homology, i.e. about 39% identity, with a chitinase protein precursor from Apha~2odidium album. (GenBank accession number P32470). Nucleic acid sequence SEQ ID N0:43 showed no significant homology to any of the sequences submitted to GenB ank.

Example 11 This example describes the isolation of nucleic acid molecules encoding DernzatopTzagoides ptero>zyssius 98 kD allergen protein.
Nucleic acid molecules with high homology to the D. farinae 98 kD allergen (map B) were isolated from a D. ptero~zyssius cDNA library by hybridization with a 32-P labeled cDNA encoding the D. fari>zae HMW-map composition.
A D. pteronyssius cDNA library was prepared as follows. Total RNA was extracted from approximately 2 grams of D. pterorzyssius mites, using an acid-guanidium-phenol-chloroform method, described by Chomzynski et al., 1987, Aizal.
Biochem 162: pp 156-159. Poly A+ selected RNA was separated from the total RNA
preparation by oligo-dT cellulose chromatography using the mRNA Purification Kit (available from Pharmacia, Newark, NJ), according to the method recommended by the manufacturer. A whole D. pteronyssius cDNA library was constructed in lambda-Uni-ZAPTM XR vector (available from Stratagene, La Jolla, CA), using Stratagene's ZAP-cDNA Synthesis Kit protocol. Approximately 5 milligram (mg) of Poly A+
RNA was used to produce the D. pteronyssius cDNA library.
Using a modification of the protocol described in the cDNA Synthesis Kit (available from Stratagene), the whole D, pterorzyssius cDNA library was screened, using duplicate plaque lifts, with a 32P-labeled cDNA encoding the D. farizZae 97 kD
Map B allergen, i.e. SEQ ID N0:17. Hybridization was done in 6X SSC (for recipe see Sambrook, et al., ibid.), 5X Denhardt's solution (for recipe see Sambrook, et al., ibid.), 0.5% sodium dodecyl sulfate (SDS) (available from Sigma), and 100 mglml of single stranded DNA (available from Sigma), at 55°C, for about 36 hours. The filters were washed 3 times, for about 30 minutes per wash, at 55°C, in 2X SSC, 0.2% SDS, followed by a final wash of about 30 minutes, at 55°C, in 0.2X SSC, 0.2°lo SDS. A
plaque purifiedclone of the D. pteroyzyssius nucleic acid molecule encoding the D.
pteroTZyssius 97 kD allergen (map B) was converted into a double stranded recombinant molecule using the ExAssist TM helper phage and SOLRTM E. coli according to the ih vivo excision protocol described in the ZAP-cDNA Synthesis Kit (all available from Stratagene). The plasmid containing the D. pterotzyssius clone was subjected to DNA sequence analysis using standard techniques. DNA sequence analysis, including the determination of molecular weight and isoelectric point (pI) was performed using the GCGTM program.
A clone was isolated that included an about 1621-nucleotide insert, which includes the full-length coding region, referred to herein as nDerp981~2i, with a coding strand represented as SEQ ll~ N0:34 and a complementary strand represented as SEQ
ID N0:36. The apparent start and stop codons span from nucleotide 14 through nucleotide 16, and from nucleotide 1541 through nucleotide 1543, respectively, of SEQ ID N0:34. A putative polyadenylation signal (5' AATAAA 3' ) is located in a region spanning from nucleotide 1584 to 1589 of SEQ ID N0:34.
Translation of SEQ ID N0:34 yields a protein of about 509 amino acids, denoted PDerp985o9, the amino acid sequence of which is presented as SEQ ID
N0:35.
The nucleic acid molecule consisting of the coding region encoding PDerp985o~
is referred to herein as nDerp981sz~, the nucleic acid sequence of which is represented as SEQ ID N0:37 (the coding strand), and SEQ ID NO:39 (the complementary strand).
The amino acid sequence of PDerp985o9, also represented herein as SEQ ID
N0:38, has an estimated molecular weight of about 58.9 kD and an estimated pI of about 5.61.
Analysis of PDerp985os suggests the presence of a signal peptide spanning from about amino acid 1 through about amino acid 19. The proposed mature protein, denoted herein as PDerp9849o, contains about 490 amino acids, and is represented herein as SEQ ID NO:41. The amino acid sequence of PDerp98~~o predicts the protein to have an estimated molecular weight of about 56.8 kD, and an estimated pI of about 5.49, as well as two asparagine-linked glycosylation sites extending from about amino acid 115 to about amino acid 117, and extending from about amino acid 240 to amino acid 242, respectively. The nucleic acid molecule encoding PDerp9849o is known as nDerp981ø~0, with a coding strand represented by SEQ ID N0:40 and a complementary strand represented by SEQ ID N0:42.
A BLAST search was performed as described previously. PDerp985o9, SEQ ID
N0:35, showed the highest homology at the amino acid level with the Mazzduca sexta chitinase (SwissProt accession number p36362), with about a 34% identity.
nDerp9816au SEQ ID N0:34, showed the highest homology at the nucleic acid level to Chelorzus sp. chitinase (accession number U10422), with about a 49% identity.

Comparison of cDNA regions corresponding to the coding regions for the D.
faf~iuae 98 kD allergen protein and the cDNA regions corresponding to the coding regions for the D. pteroiZyssius 98 kD allergen protein shows an identity of about 84%.
Example 14.
This example demonstrates the binding of the D. farifZae HMW-map composition to human IgE in human sera isolated from humans known to be allergic to mite allergens.
A technique called RAST, or radio-allergo-absorbent test, was used because the amount of human IgE present in human sera is quite low. RAST was essentially performed as described in Aalberse, RC et al., (1981) J. Allef~gy CliiZ
In2mma. 68: pp 356-364. To calculate the unit IU/ml, 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) JAllergy Clih ImnZUn.ol. 99: pp 545-550).
Briefly, 50 ,ug of the HMW-map composition, purified as described in Example 1, was coupled to 50 mg of CNBr-activated Sepharose 4B (available from Pharmacia, Piscataway, NJ), according to the manufacturer's protocols. Human sera were selected (17 different samples, total) on the basis of a positive RAST
for whole mite D. fariuae extracts, a positive RAST number is greater than 1 IU/ml). Two negative (less than 0.3 IU) control sera were also included.
To test each individual serum sample, 0.5 mg of the D. fariJ2ae HMW-map composition-coupled Sepharose was incubated with 50 ,u1 serum in a total volume of 300 ,u1 of PBS-T (Phosphate buffered saline with added 0.1% volume/volume Tween-20, available from Sigma). Incubation was overnight at 27°C, with shaking. After incubation, the coupled Sepharose was washed five times with PBS-T.
Radiolabelled (ias-Iodine) sheep anti-human IgE, made by standard radioiodination protocols, (diluted in PBS-T with 4.5% bovine serum and 0.5% sheep serum, v/v) in a total volume of 750 ,u1, was added and incubated overnight at 27°C. After incubation, the coupled Sepharose was washed four times with PBS-T and counted in a gamma-counter to determine the amount of radiolabeled sheep anti-human IgE bound to the HMW-map composition-coupled Sepharose. The results are shown in Table 4.

_88_ Table 4. Binding of human IgE to HMW-map composition from D. farizzae Serum number RAST, D: farinae RAST, HMW-map whole comps'n., IU
extract, IU

1445 > 100 48 1456 >100 42 1458 21.1 0.5 1460 14.1 2.5 1463 37.6 0.1 1464 37.2 2.0 1465 14.5 0.7 1466 89.9 7.7 1468 >100 19.9 1471 31.9 0.8 1491 23.8 1.0 1496 25.3 3.6 1505 5.1 0.2 1523 1.0 <0.1 1529 1.2 0.7 1530 (control)0.2 <0.1 1531 (control)0.1 <0.1 Almost 75% of patients (11 of 15) who showed sensitivity to D. farizzae whole mite extracts were sensitive to the HMW-map composition antigen, implying that the HMW-map composition antigen is a major antigen for D. farirzae sensitive humans.
Sensitivity to the HMW-map composition was defined as a RAST of greater than or equal to 0.5 ICT.
Example 15.
This example demonstrates that the D. farizzae HMW-map composition described in Example 1 includes a glycoprotein.
About (5.4 ,ug) of a D. farirzae HMW-map composition prepared in accordance with Example 1 was applied to SDS PAGE and electrophoresis was done according to standard techniques. The protein was blotted to a nitrocellulose membrane according to standard techniques, and glycoprotein was detected using the DIGT"~ Glycine Detection Kit (available from Boehringer Mannheim, Indianapolis, IN), using the manufacturer's protocol. The region corresponding to the HMW-map region showed a positive reaction with the kit, indicating that the HMW-map composition includes a glycoprotein.
Example 16.
This example shows that the D. faritaae HMW-map composition retains its character as an allergen even when the amino acid residues are removed, both by chemical and enzymatic means. The results suggest that the main epitope(s) could be a carbohydrate epitope including a polysaccharide attached to an N-linked or O-linked glycosylation site on the HMW-map composition.
A. Protein elimination by chemical means ((3-elimination of proteins) Twelve ,ug (microgram) of HMW-map composition (purified as described in Example 1) was dissolved in 100 ,u1 (microliter) of distilled deionized water.
To this mixture was added 5 ,u1 10 M (molar) NaOH and 3.8 mg (milligram) NaBH4 (available from Sigma) to give a final concentration of 0.5 M NaOH and 1 M NaBH4. This reaction mixture was heated at 50°C for 30 minutes, then cooled, and 100 ,u1 acetone was added. To this mixture, sufficient amount, i.e. approximately 150 ,u1, of Dowex 50 (H+) (available from Pharmacia) was added to make the solution slightly acidic.
The Dowex 50 adsorbed and removed the protein, leaving any sugar moieties in the supernatant. The mixture was centrifuged in a microcentrifuge and washed three times with 100 ,u1 of water. The combined supernatants from the centrifugations were evaporated to dryness, then washed five times from a methanol:HCl solution (1000:1 v/v), evaporating to dryness after each wash, to remove salts. The mixture was dissolved in 100 ,u1 of water, and a portion (20 ,u1) was analyzed by SDS-PAGE
using standard techniques, and both Coomassie blue and Silver staining were used to determine the amount of protein in the chemically treated samples. No protein was detected by either Coomassie or Silver staining, indicating removal of protein. Any sugar moieties on the protein would be unaffected by these conditions.
The remainder of the residue from each sample was subjected to ELISA
analysis as described in Example 4. Briefly, 100 ng of either the (3-eliminated sample or of non-(3-eliminated sample of the HMW-map composition was coated onto the Immulon plates, and ELISAs were carried out as described in Example 4 with a D.
farircae sensitive dog sera pool, a D. farahae sensitive cat sera pool, and various individual dog sera that are either D. farihae sensitive or not sensitive (as measured by ELISA). The results are shown in Table 5.
Table 5. Reactivity of dog and cat sera to HMW-map composition and to (3-eliminated HMW-map composition (which is carbohydrate only) Sera used .. (3-eliminated HMW-map;untreated I3MW-map .. comps'n., OD X
OD (carbohydrate 10'3 antigen) D.farinae dog 1233 1931 pool D. farihae cat 2837 3115 pool dog 1621A 15 0 dog 1621C 24 21 dog 1621 S 59 420 dog 1626C 23 214 dog SPF-2 16 0 Results from Table 5 indicate that the (3-eliminated HMW-map composition sample still retains the ability to bind IgE from dog and cat sera that is sensitive to D. fari~2ae HMW-map composition, indicating that the glycans attached to the protein constitute a major epitope of the HMW-map composition allergen protein.
B. Protein Elimination by enzymatic means.
14 ,ug of HMW-map composition (purified as described in Example 1) was digested with l,ug Endoproteinase K, available from Sigma, to remove the protein moiety of the molecule. The digestion reaction took place at 56°C for 24 hours, after which the endoproteinase in the reaction was heat-denatured in boiling water for 10 minutes.
A portion of this reaction was analyzed by SDS-PAGE using standard techniques, and both Coomassie blue and Silver staining were used to detect the presence of protein in the enzymatically digested samples. No HMW-map composition was detected by either Coomassie or Silver staining, indicating elimination of the HMW-map composition. Any glycan that was attached via a glycosylation site on the protein would be unaffected by these conditions.

The remainder of the enzymatically digested reaction was tested by ELISA in the manner described in Example 4. Briefly, 100 ng of either the proteinase-K-digested sample or of a non-digested sample of the HMW-map composition was coated onto Immulon plates, and ELISAs were carried out as described in Example 4 with various individual dog sera that were either D. fari~zae sensitive or not sensitive (as measured by ELISA). The results are shown in Table 6.
Table 6. Reactivity of dog sera to HMW-map composition and to Endoproteinase-K digested HMW-map composition.
dog D. farir2ae OD, wells chafed OD; wells coated with # sensitive?1 with Proteinase K
HMW-map, cotnps'n. digested HMW-map :, , 1 yes 120 122 2 yes 1637 1561 3 yes 858 383 4 yes 914 509 5 yes 277 227 6 yes 2891 2636 7 no 10 11 8 yes 4056 3880 9 yes 1920 1626 10 yes 472 432 11 yes 328 213 12 yes 2913 2530 13 yes 1232 984 14 yes 3153 2355 15 no 6 46 16 yes 860 339 17 yes 2429 750 18 yes 1194 351 19 yes 2655 1443 20 yes 3285 1207 0 21 yes 2636 1240 22 yes 1097 848 23 yes 1621 1408 24 yes 2113 1592 25 yes 1169 408 26 yes 4200 4200 27. yes 4200 4200 dog D. farinae OD, wells coated OD, wells coated with # sensitive?1 with Pxoteinase K
HMW-map comps'n. digested HMW-map 28 yes 3222 2932 29 yes 2468 2118 30 yes 3339 2454 31 no 0 4 ' by ELISA in a separate experiment Results from Table 6 indicate that the proteinase-I~ digested HMW-map composition sample still retains the ability to bind IgE from dog and cat sera that is sensitive to D.
farihae HMW-map composition, suggesting that the glycans attached to the protein constitute a major epitope on the HMW-map composition.
Example 17 This example describes attempts to remove N-linked glycans from the HMW-map composition.
HMW-map composition (2 ,ug), purified as in Example 1, was digested with N-glycosidase F (available from Boehringer-Mannheim), according to the manufacturer's directions. The digestion was analyzed by SDS-PAGE and stained according to standard protocols. 2 ,ug Fetuin (available from Sigma) was used as a positive N-linked glycosylated protein control. Analysis of the SDS-PAGE showed that there were no apparent differences in the molecular weights of the intact and digested map B
protein. The positive control, fetuin, did show a reduction of molecular weight after digestion with N-glycosidase F. This result indicates that there are no N-linked glycans on the HMW-map composition, or alternatively that there are only small sized N-glycans on the HMW-map composition.
Example 18 This example describes the isolation and sequencing of a nucleic acid molecule encoding the full length Dermatophagoides fariyzae 60 kD allergen.
This nucleic acid molecule was isolated from a Derfvatophagoides farircae cDNA library by it's ability to hybridize with a 32P-labeled cDNA encoding a portion of the Dermatoplzagoides fararcae 60 kD allergen described in Example 10.
A Dermatophagoides faf°inae cDNA library was prepared as follows.
Total RNA was extracted from approximately 2 grams of D. farir~ae. mites, using an acid-guanidinium-phenol-chloroform method similar to that described by Chomzynski et al., 1987, Anal. Biochern. 162,156-159. Poly A+ selected RNA was separated from the total RNA preparation by oligo-dT cellulose chromatography using the mRNA
Purification Kit (available from Pharmacia Biotech, Newark, NJ), according to the method recommended by the manufacturer. A whole mite cDNA library was constructed in lambda-Uni-ZAPS XR vector (available from Stratagene), using Stratagene's ZAP-cDNA Synthesis Kit protocol. Approximately 5 ~g of Poly A+
RNA
was used to produce the D. farinae cDNA library.
Using a modification of the protocol described in the cDNA Synthesis Kit, the whole mite cDNA library was screened, using duplicate plaque lifts, with 32P-labeled cDNA nDerf605io. Hybridization was done at 6XSSC, 5X Denhardt's solutions, 0.5 %
SDS, 100 mg/ml of ssDNA and, at 52°C, for 18 hr. The filters were washed 2 times, for 30 minutes per wash, at 55° C in 2XSSC, 0.2°7o SDS, followed by a final wash of 30 minutes in the same buffer except using aboutØ2XSSC: A plaque purified clone of the nucleic acid molecules encoding the D. farinae 60 kD allergen was converted into a double stranded recombinant molecule, herein denoted as nDerf601ass , using the ExAssist~ 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.
Example 19 This example describes the sequencing of a D. farinae nucleic acid molecule of the present invention.
The plasmid containing nDerf6014ss was sequenced by the Sanger dideoxy chain termination method, using the PRISMS Ready Dye Terminator Cycle Sequencing Kit with Ampli Taq DNA Polymerase, FS (available from the Perkin-Elmer Corporation, Norwalk, CT). PCR extensions were done in the GeneAmpTM PCR
System 9600 (available from Perkin-Elmer). Excess dye terminators were removed from extension products using the CentriflexTM Gel Filtration Cartridge (available from Advanced Genetics Technologies Corporation, Gaithersburg, MD) following the manufacturer's standard protocol. Samples were resuspended according to ABI

protocols and were run on a Perkin-Elmer ABI PRISM TM 377 Automated DNA
Sequencer. DNA sequence analysis, including the compilation of sequences and the determination of open reading frames, was performed using the GCGTM program (available from Genetics Computer Group, Madison, WI). Protein sequence analysis, including the determination of molecular weight and isoelectric point (pI) was performed using the GCG~ program.
An about 1455 nucleotide consensus sequence of the entire nDerf6014ss nucleic acid molecule was determined; the sequences of the two complementary strands are presented as SEQ ID N0:50 (the coding strand) and SEQ ID NO: 52 (the complementary strand). The nDerf6014ss sequence contains a full length coding region.
The apparent start and stop codons span nucleotides from 14 through 16 and from 1400 through1402, respectively, of SEQ ID NO: 50. A putative polyadenylation signal (5' AATAAA 3' ) is located in a region spanning from about nucleotide 1413 of SEQ ID NO: 50.
Translation of SEQ ID NO: 50 yields a protein of 462 amino acids, denoted PDerf6046z, the amino acid sequence of which is presented in SEQ ID NO: 51.
The nucleic acid molecule consisting of the coding region encoding PDerf60d~z is referred to herein as nDerf6013s6, the nucleic acid sequence of which is represented in SEQ ID
NO: 53 (the coding strand) and SEQ ID NO: 54 (the complementary strand). The amino acid sequence of PDerf604~z (i.e., SEQ ID NO: 51) predicts that PDerf604~z has an estimated molecular weight of about 52.1 kD and an estimated pI of about 5.73.
Analysis of SEQ ID 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 PDerf6043~, contains about 437 amino acids which is represented herein as SEQ ID NO: 56. The amino acid sequence of PDerf6043~ (i.e., SEQ ID NO: 56) predicts that PDerf60ø6z 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. The nucleic acid molecule encoding the mature protein is denoted SEQ ID NO: 55 and its reverse complement is denoted SE ID NO: 57.

A BLASTp search was performed according to Altschul, et al, (1990), J. Mol.
Biol. 215:403-410; and Altschul, et al, (1997), Nucleic Acids Res. 25:3389-3402. The protein search was performed using SEQ ID N0:51, which showed significant homology to chitinase molecules. The highest scoring match of the homology search at the amino acid level was PIR accession number A53918: Cheloi2us sp.
chitinase precursor, which was about 32°1o identical with SEQ ID N0:51. At the nucleotide level, the search was performed using SEQ ID N0:53, which did not show significant similarity to any sequences in the database. Sequence analysis was performed using the GCG GAP program as described above.
Example 20 This example further describes the characterization of the D. farihae HMW-map composition (also referred to as Der f 15).
Nucleic acid molecule nDerf981~s2 of Example 8 was inserted into appropriate expression vectors and expressed in E. coli and P. pastoris. When the resulting protein, PDerf98sss 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. farihae 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.
All of these results together suggest that the main epitope(s) are carbohydrate regions of the molecule or some other secondary modification.
The antigenicity of the native Der f 15 protein is not lost after periodate treatment; generally carbohydrate epitopes are destroyed by periodate except for those further substituted with additional groups or those having an unusual sugar with no geminal hydroxyl groups.
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 GIcNAc and GalNAc.
The native Der f 15 protein was treated with base in the presence of NaBH4 and analyzed by a P-4 sizing chromatography. O-linked oligosaccharides present in Der f were found to void the column. This result is consistent with either very large O-linked oligosaccharides or the presence of acidic groups on the oligosaccharides such as sulfate. Attempts to determine the presence or absence of sulfate more directly gave 10 ambiguous results.
Der f 15 was treated at pH 4, pH 5, and pH 7 overnight at 37° C. The resulting samples were then probed with antibody to the protein or dog serum known to be reactive with Der f 15. In the samples treated at pH 5 and pH 7, all of the dog antiserum epitope was destroyed, but in the samples treated at pH 4, some activity 15 remained. The anti-Der f 15 antibody shows that the molecular weight of Der f 15 was decreased at all pH's with some original material left at pH 4, as though deglycosylation was occurring. It is not known whether this change was self catalyzed by the Der f 15 protein or occurred chemically; while not being bound by theory, it is believed that self catalysis was involved since the loss of the epitope occurred under such mild conditions.
Example 21 This example describes the binding of several house dust mite (HDM) allergens to feline IgE in cat serum.
The allergen profile of the IgE response of cats to house dust mites appears to be different fiom that of dogs. An examination of the results of IgE testing on cat sera submitted to Heska's Veterinary Diagnostic Laboratories (VDL) in January 2000 shows that 40% of all allergen-specific IgE positive cats had anti-HDM IgE.
All the cats were positive to both D. fari~ae and D. pterofzyssiyaus. Eighty-eight sera known to be positive for D. farinae were assayed by ELISA on highly purified preparations of Der f 1, Der f 2, Der f 15, and the 60 kD allergen. In this assay, 32% of the cats were positive for Der f l, 42% were positive for Der f 2, 68% were positive for Der f 15, and 86% were positive for the 60 kD allergen.
While various embodiments of the present invention have been described in detail, it is apparent that modifications and adaptations of those embodiments will occur to those skilled in the art. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention;
as set forth in the following claims.

AL-2-C4-PCT.ST25.txt SEQUENCE LISTING
<110> Heska Corporation MCCall, Catherine A.
Hunter, Shirley Wu Weber, Eric R.
<120> NOVEL DERMATOPHAGOIDES NUCLEIC ACID MOLECULES, PROTEINS AND USES
THEREOF
<130> AL-2-C4-PCT
<140> not yet assigned <141> 2001-09-13 <150> 09/662,293 <151> 2000-09-14 <150> 09/292,225 <151> 1999-04-15 <150> 60/098,909 <151> 1998-09-02 <150> 60/085,295 <151> 1998-05-13 <150> 6.0/098,565 <151> 1998-04-17 <150> 09/062,013 <151> 1998-04-17 <160> 57 <170> Patentln version 3.1 <210> 1 <211> 14 <212> PRT
<213> Dermatophagoides farinae <400> 1 Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met <210> 2 <211> 20 <212> PRT
<213> Dermatophagoides farinae <400> 2 Asp Tyr Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ala Pro Leu Tyr Lys Arg Pro <210> 3 AL-2-C4-PCT.ST25.txt <211> 20 <212> PRT
<213> Dermatophagoides farinae <400> 3 Asp Ile Pro His Pro Thr Asn Ile His Lys Tyr Leu Val Cys Glu Ser Val Asn Gly Gly <210> 4 <211> 20 <212> PRT
<213> Dermatophagoides farinae <400> 4 Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Val Gly Glu Glu Gly Val Leu Ser <210> 5 <211> 12 <212>. PRT
<213> Dermatophagoides farinae <400> 5 Asp Glu Lys Asn Ser Phe Glu Cys Ile Leu Gly Pro <210> 6 <211> 18 <212> PRT
<213> Dermatophagoides farinae <400> 6 Asp Ala Phe Glu Pro His Gly Tyr Leu Leu Thr A1a Ala Val Ser Pro Gly Lys <210> 7 <211> 13 <212> PRT
<213> Dermatophagoides farinae <400> 7 Asp Lys Gln Asn Tyr Leu Ala Leu Val Arg Glu Leu Lys AL-2-C4-PCT.ST25.txt <210> 8 <211> 24 <212> PRT
<213> Dermatophagoides farinae <400> 8 Asp Met Ala Gln Asn Tyr Lys Tyr Arg Gln Gln Phe Ile Gln Ser Val Leu Asn Asn Gly Ala Thr Arg Gln <210> 9 <211> 23 <212> PRT
<213> Dermatophagoides farinae <220>
<221> MISC_FEATURE
<222> (3). (3) <223> Xaa = any amino acid at position 3 <220>
<221> MISC_FEATURE
<222> (7). (7) <223> Xaa = any amino acid at position 7 <400> 9 Asp Glu Xaa Asn Val Met Xaa Tyr Val Leu Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg <210> 10 <211> 17 <212> PRT
<213> Dermatophagoides farinae <220>
<221> MISC_FEATURE
<222> (14) .(14) <223> Xaa = any amino acid at position 14 <400> 10 Asp Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Xaa Ser Ile Glu AL-2-C4-PCT. ST25 . tact <210> 11 <211> 19 <212> PRT
<213> Dermatophagoides farinae <400> 11 Asp Ile Pro His Pro Thr Asn Ile His Lys Tyr Leu Val Cys Glu Ser Val Asn Gly <210> 12 <211> 18 <212> PRT
<213> Dermatophagoides farinae <400> 12 Asp Tyr Ala Lys Asn Pro Lys Arg I1e Val Cys Ile Val Gly Thr Glu Gly Val <210> 13 <211> 20 <212> PRT
<213> Dermatophagoides farinae <400> 13 Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Val Gly Glu Glu G1y Val Leu Ser <210> 14 <211> 1752 <212> DNA
<213> Dermatophagoides farinae <220>
<221> CDS
<222> (1)..(1665) <223>
<400> 14 atg aaa acc ata tat gca ata ctt agt att atg gcc tgc att ggc ctt 48 Met Lys Thr Ile Tyr Ala Ile Leu Ser Ile Met Ala Cys Ile Gly Leu atg aat gca tcc atc aaa cga gat cat aat gat tat tcg aaa aat ccg 96 Met Asn Ala Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro AL-2-C4-PCT.ST25.txt atgaga attgtt tgttatgttgga acatggtcc gtatatcat aaagtt 144 MetArg IleVal CysTyrVa1Gly ThrTrpSer ValTyrHis LysVal gatcca tacact atcgaagatatt gatccattc aagtgtaca cattta 192 AspPro TyrThr IleGluAspIle AspProPhe LysCysThr HisLeu atgtat ggtttc getaaaattgat gaatacaaa tacacaatt caagtt 240 MetTyr GlyPhe AlaLysIleAsp GluTyrLys TyrThrIle GlnVal ttcgat ccttac caagatgataac cataactca tgggaaaaa cgtggt 288 PheAsp ProTyr GlnAspAspAsn HisAsnSer TrpGluLys ArgGly tatgaa cgtttc aacaacttgcga ttgaagaat ccagaatta accacc 336 TyrGlu ArgPhe AsnAsnLeuArg LeuLysAsn ProGluLeu ThrThr atgatt tcactt ggtggttggtat gaaggctcg gaaaaatat tccgat 384 MetIle SerLeu GlyGlyTrpTyr GluGlySer GluLysTyr SerAsp atgget gcaaat ccaacatatcgt caacaattc atacaatca gttttg 432 MetAla AlaAsn ,ProThrTyrArg GlnGlnPhe IleGlnSer ValLeu gacttt ttgcaa gaatacaagttc gacggtcta gatttggat tgggag 480 AspPhe LeuGln GluTyrLysPhe AspGlyLeu AspLeuAsp TrpGlu tatcct ggatct cgattgggtaac ccgaaaatc gataaacaa aactat 528 TyrPro GlySer ArgLeuGlyAsn ProLysIle AspLysGln AsnTyr ttgget ttggtt agagaacttaaa gacgetttt gaacctcat ggctac 576 LeuAla LeuVal ArgGluLeuLys AspAlaPhe GluProHis GlyTyr ttgttg actget gcagtatcacca ggtaaagac aaaatcgac cgaget 624 LeuLeu ThrAla AlaValSerPro GlyLysAsp LysIleAsp ArgAla tatgat atcaaa gaattgaacaaa ttgttcgat tggatgaat gtcatg 672 TyrAsp IleLys GluLeuAsnLys LeuPheAsp TrpMetAsn ValMet acatat gattac cacggtggatgg gaaaacttt tacggtcac aatget 720 ThrTyr AspTyr HisGlyGlyTrp GluAsnPhe TyrGlyHis AsnAla ccgttg tataaa cgaccagatgaa actgatgag ttgcacact tacttc 768 ProLeu TyrLys ArgProAspGlu ThrAspGlu LeuHisThr TyrPhe aatgtc aactac accatgCactat tatttgaac aatggtgcc accaga 816 AsnVal AsnTyr ThrMetHisTyr TyrLeuAsn AsnGlyAla ThrArg gac aaa ttg gta atg ggt gtt cca ttc tat ggc cgt get tgg agc att 864 Asp Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser Ile AL-2-C4-PCT.ST25.txt gaa gatcgaagc aaactcaaa ctt-ggagat ccagccaaa ggcatgtcg 912 Glu AspArgSer LysLeuLys LeuGlyAsp ProAlaLys GlyMetSer CCC CCaggtttc atttctggt gaagaaggt gtcctctca tatatagaa 960 Pro ProGlyPhe IleSerGly GluGluGly ValLeuSer TyrIleGlu ~

ttg tgtcaattg tttcaaaaa gaagaatgg catatccaa tacgatgaa 1008 Leu CysGlnLeu PheGlnLys GluGluTrp HisIleGln TyrAspGlu tat tacaatget ccatatggt tacaatgat aaaatctgg gtcggttac 1056 Tyr TyrAsnAla ProTyrGly TyrAsnAsp LysIleTrp ValGlyTyr gatgatctggcc agtata tcatgcaagttg getttcctg aaagaatta 1104 AspAspLeuAla SerIle SerCysLysLeu AlaPheLeu LysGluLeu ggcgtttctggt gtcatg gtttggtcattg gaaaatgat gatttcaaa 1152 GlyValSerGly ValMet ValTrpSerLeu GluAsnAsp AspPheLys ggtcactgcgga ccgaaa aatccattgttg aacaaagtt cataatatg 1200 GlyHisCysGly ProLys AsnProLeuLeu AsnLysVal HisAsnMet attaatggcgat gaaaag aactctttcgaa tgcattttg ggtccaagt 1248 IleAsnGlyAsp GluLys AsnSerPheGlu CysIleLeu GlyProSer acaacgacacca actcca acgacgacaccc acaaccccg actacaacg 1296 ThrThrThrPro ThrPro ThrThrThrPro ThrThrPro ThrThrThr ccaacaactcct tctccc accaccccgaca acaacccct tctcccacc 1344 ProThrThrPro SerPro ThrThrProThr ThrThrPro SerProThr accccgacaaca acccct tctcccaccaca ccgacaaca actccttct 1392 ThrProThrThr ThrPro SerProThrThr ProThrThr ThrProSer cccaccacacca acacca acaacaccaaca ccagcccct acaacatcg 1440 ProThrThrPro ThrPro ThrThrProThr ProAlaPro ThrThrSer acaccttcgcca accacg accgaacacaca agcgaaaca ccaaaatat 1488 ThrProSerPro ThrThr ThrG1uHisThr SerGluThr ProLysTyr acaacctatgtc gatgga catcttatcaaa tgttacaag gaaggtgat 1536 ThrThrTyrVal AspGly HisLeuIleLys CysTyrLys GluGlyAsp atcccacatcca accaat atacacaaatat ttggtctgt gaatttgtt 1584 IleProHisPro ThrAsn IleHisLysTyr LeuValCys GluPheVal aatggtggctgg tgggtt catattatgccc tgtccaccg ggcactatt 1632 AsnGlyGlyTrp TrpVal HisIleMetPro CysProPro GlyThrIle AL-2-C4-PCT.ST25.txt tgg tgt caa gaa aaa ttg act tgt ata ggc gaa taattctgaa aaaaaaattc 1685 Trp Cys Gln Glu Lys Leu Thr Cys Ile Gly Glu aattaaaatt taaaattcaa tttttaatat gaaaaattca aaaaaaaaaa aaaaaaaaaa 1745 aaaaaaa 1752 <210> 15 <211> 555 <212> PRT
<213> Dermatophagoides farinae <400> 15 Met Lys Thr Ile Tyr Ala Ile Leu Ser Ile Met Ala Cys Ile Gly Leu Met Asn Ala Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met Arg Ile Val Cys Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val Asp Pro Tyr Thr Ile Glu Asp Ile Asp Pro Phe Lys Cys Thr His Leu Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val Phe Asp Pro Tyr Gln Asp Asp Asn His Asn Ser Trp Glu Lys Arg Gly Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Ile Gln Ser Val Leu Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln Asn Tyr Leu Ala Leu Val Arg Glu Leu Lys Asp Ala Phe Glu Pro His Gly Tyr AL-2-C4-PCT.ST25.txt Leu Leu Thr Ala Al.a Val Ser Pro Gly Lys Asp Lys I1e Asp Arg Ala Tyr Asp Ile Lys Glu Leu Asn Lys Leu Phe Asp Trp Met Asn Val Met Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn Phe Tyr Gly His Asn Ala Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr Tyr Phe Asn Val Asn Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg Asp Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser Ile Glu Asp Arg Ser Lys Leu Lys Leu Gly Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Ser Gly G1u Glu Gly Val Leu Ser Tyr Ile Glu Leu Cys Gln Leu Phe Gln Lys Glu G1u Trp His Ile Gln Tyr Asp G1u Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr Asp Asp Leu Ala Ser Ile Ser Cys Lys Leu Ala Phe Leu Lys Glu Leu Gly Val Ser Gly Val Met Val Trp Ser Leu Glu Asn Asp Asp Phe Lys Gly His.Cys Gly Pro Lys Asn Pro Leu Leu Asn Lys Val His Asn Met Ile Asn Gly Asp Glu Lys Asn Ser Phe Glu Cys Ile Leu Gly Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr Pro Thr Thr Pro Thr Thr Thr Pro Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr AL-2-C4-PCT.ST25.txt Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr Ser Thr Pro Ser Pro Thr Thr Thr Glu His Thr Ser Glu Thr Pro Lys Tyr Thr Thr Tyr Val Asp Gly His Leu Ile Lys Cys Tyr Lys Glu Gly Asp Ile Pro His Pro Thr Asn Ile His Lys Tyr Leu Val Cys Glu Phe Val Asn Gly Gly Trp Trp Val His Ile Met Pro Cys Pro Pro Gly Thr Ile Trp Cys Gln Glu Lys Leu Thr Cys Ile Gly Glu <210>

<211>

<212>
DNA

<213>
Dermatophagoides farinae <400>

ttttttttttttttttttttttttttttgaatttttcatattaaaaattgaattttaaat60 tttaattgaatttttttttcagaattattcgcctatacaagtcaatttttcttgacacca120 aatagtgcccggtggacagggcataatatgaacccaccagccaccattaacaaattcaca180 gaccaaatatttgtgtatattggttggatgtgggatatcaccttccttgtaacatttgat240 aagatgtccatcgacataggttgtatattttggtgtttcgcttgtgtgttcggtcgtggt300 tggcgaaggtgtcgatgttgtaggggctggtgttggtgttgttggtgttggtgtggtggg360 agaaggagttgttgtcggtgtggtgggagaaggggttgttgtcggggtggtgggagaagg420 ggttgttgtcggggtggtgggagaaggagttgttggcgttgtagtcggggttgtgggtgt480 cgtcgttggagttggtgtcgttgtacttggacccaaaatgcattcgaaagagttcttttc540 atcgccattaatcatattatgaactttgttcaacaatggatttttcggtccgcagtgacc600 tttgaaatcatcattttccaatgaccaaaccatgacaccagaaacgcctaattctttcag660 gaaagccaacttgcatgatatactggccagatcatcgtaaccgacccagattttatcatt720 gtaaccatatggagcattgtaatattcatcgtattggatatgccattcttctttttgaaa780 caattgacacaattctatatatgagaggacaccttcttcaccagaaatgaaacctggggg840 AL-2-C4-PCT.ST25.txt cgacatgcctttggctggatctccaagtttgagtttgcttcgatcttcaatgctccaagc900 acggccatagaatggaacacccattaccaatttgtctctggtggcaccattgttcaaata960 atagtgcatggtgtagttgacattgaagtaagtgtgcaactcatcagtttcatctggtcg1020 tttatacaacggagcattgtgaccgtaaaagttttcccat.ccaccgtggtaatcatatgt1080 catgacattcatccaatcgaacaatttgttcaattctttgatatcataagctcggtcgat1140 tttgtctttacctggtgatactgcagcagtcaacaagtagccatgaggttcaaaagcgtc1200 tttaagttctctaaccaaagccaaatagttttgtttatcgattttcgggttacccaatcg1260 agatccaggatactcccaatccaaatctagaccgtcgaacttgtattcttgcaaaaagtc1320 caaaactgattgtatgaattgttgacgatatgttggatttgcagccatatcggaatattt1380 ttccgagccttcataccaaccaccaagtgaaatcatggtggttaattctggattcttcaa1440 tcgcaagttgttgaaacgttcataaccacgtttttcccatgagttatggttatcatcttg1500 gtaaggatcgaaaacttgaattgtgtatttgtattcatcaattttagogaaaccatacat1560 taaatgtgtacacttgaatggatcaatatcttcgatagtgtatggatcaactttatgata1620 tacggaccatgttccaacataacaaacaattctcatcggatttttcgaataatcattatg1680 atctcgtttgatggatgcattcataaggccaatgcaggccataatactaagtattgcata1740 tatggttttcat 1752 <210> 17 <211> 1665 <212> DNA
<213> Dermatophagoides farinae <220>
<221> CDS
<222> (1)..(1665) <223>
<400>

atgaaa accatatat gcaata cttagtatt atggcctgcatt ggcctt 48 MetLys ThrIleTyr AlaIle LeuSerIle MetAlaCysIle GlyLeu atgaat gcatccatc aaacga gatcataat gattattcgaaa aatccg 96 MetAsn AlaSerIle LysArg AspHisAsn AspTyrSerLys AsnPro atgaga attgtttgt tatgtt ggaacatgg tccgtatatcat aaagtt 144 MetArg IleValCys TyrVal G1yThrTrp SerValTyrHis LysVal gatcca tacactatc gaagat attgatcca ttcaagtgtaca cattta 192 AspPro TyrThrIle GluAsp IleAspPro PheLysCysThr HisLeu atgtat ggtttcget aaaatt gatgaa,tac aaatacacaatt caagtt 240 MetTyr GlyPheAla LysIle AspGluTyr LysTyrThrIle GlnVal AL-2-C4-PCT.ST25.txt ttcgatccttac caagatgat aaccataac tcatgggaaaaa cgtggt 288 PheAspProTyr GlnAspAsp AsnHisAsn SerTrpGluLys ArgGly tatgaacgtttc aacaacttg cgattgaag aatccagaatta accacc 336 TyrGluArgPhe AsnAsnLeu ArgLeuLys AsnProGluLeu ThrThr atgatttcactt ggtggttgg tatgaaggc tcggaaaaatat tccgat 384 MetIleSerLeu GlyGlyTrp TyrGluGly SerGluLysTyr SerAsp atggetgcaaat ccaacatat cgtcaacaa ttcatacaatca gttttg 432 MetAlaAlaAsn ProThrTyr ArgGlnGln PheIleGlnSer ValLeu gactttttgcaa gaatacaag ttcgacggt ctagatttggat tgggag 480 AspPheLeuGln GluTyrLys PheAspGly LeuAspLeuAsp TrpGlu tatcct ggatctcga ttgggt aacccgaaa atcgataaacaa aactat 528 TyrPro GlySerArg LeuGly AsnProLys IleAspLysGln AsnTyr ttgget ttggttaga gaactt aaagacget tttgaacctcat ggctac 576 LeuAla LeuValArg GluLeu LysAspAla PheGluProHis GlyTyr ttgttg actgetgca gtatca ccaggtaaa gacaaaatcgac cgaget 624 LeuLeu ThrAlaAla ValSer ProGlyLys AspLysI1eAsp ArgAla tatgat atcaaagaa ttgaac aaattgttc gattggatgaat gtcatg 672 TyrAsp IleLysGlu LeuAsn LysLeuPhe AspTrpMetAsn ValMet acatat gattaccac ggtgga tgggaaaac ttttacggtcac aatget 720 ThrTyr AspTyrHis GlyGly TrpGluAsn PheTyrGlyHis AsnAla ccgttg tataaacga ccagat gaaactgat gagttgcacact tacttc 768 ProLeu TyrLysArg ProAsp GluThrAsp GluLeuHisThr TyrPhe aatgtc aactacacc atgcac tattatttg aacaatggtgcc accaga 816 AsnVal AsnTyrThr MetHis TyrTyrLeu AsnAsnGlyAla ThrArg gacaaa ttggtaatg ggtgtt ccattctat ggccgtgettgg agcatt 864 AspLys LeuValMet GlyVal ProPheTyr GlyArgAlaTrp SerIle gaagat cgaagcaaa ctcaaa cttggagat ccagccaaaggc atgtcg 912 GluAsp ArgSerLys LeuLys LeuGlyAsp ProAlaLysGly MetSer ccccca ggtttcatt tctggt gaagaaggt gtcctctcatat atagaa 960 ProPro GlyPheIle SerGly GluGluGly Va1LeuSerTyr IleGlu ttgtgt caattgttt caaaaa gaagaatgg catatccaatac gatgaa 1008 LeuCys GlnLeuPhe GlnLys GluGluTrp HisIleGlnTyr AspGlu 325 , 330 335 AL-2-C4-PCT.ST25.txt tattacaatget ccatatggt tacaatgataaa atctgggtc ggttac 1056 TyrTyrAsnAla ProTyrGly TyrAsnAspLys IleTrpVal GlyTyr gatgatctggcc agtatatca tgcaagttgget ttcctgaaa gaatta 1104 AspAspLeuAla SerIleSer CysLysLeuAla PheLeuLys GluLeu ggcgtttctggt gtcatggtt tggtcattggaa aatgatgat ttcaaa 1152 GlyValSerGly ValMetVa1 TrpSerLeuGlu AsnAspAsp PheLys ggtcactgcgga ccgaaaaat ccattgttgaac aaagttcat aatatg 1200 GlyHisCysGly ProLysAsn ProLeuLeuAsn LysValHis AsnMet attaatggcgat gaaaagaac tctttcgaatgc attttgggt ccaagt 1248 Ile,AsnGlyAsp GluLysAsn SerPheGluCys IleLeuGly ProSer acaacgacacca actccaacg acgacacccaca accccgact acaacg 1296 ThrThrThrPro ThrProThr ThrThrProThr ThrProThr ThrThr ccaacaactcct tctcccacc accccgacaaca accccttct cccacc 1344 ProThrThrPro SerProThr ThrProThrThr ThrProSer ProThr accccgacaaca accccttct CCCaCCaCaCCg acaacaact ccttct 1392 ThrProThrThr ThrProSer ProThrThrPro ThrThrThr ProSer cccaccacacca acaccaaca acaccaacacca gcccctaca acatcg 1440 ProThrThrPro ThrProThr ThrProThrPro AlaProThr ThrSer acaccttcgcca accacgacc gaacacacaagc gaaacacca aaatat 1488 ThrProSerPro ThrThrThr GluHisThrSer GluThrPro LysTyr acaacctatgtc gatggacat cttatcaaatgt tacaaggaa ggtgat 1536 ThrThrTyrVal AspGlyHis LeuIleLysCys TyrLysGlu GlyAsp atcccacatcca accaatata cacaaatatttg gtctgtgaa tttgtt 1584 IleProHisPro ThrAsnIle HisLysTyrLeu ValCysGlu PheVal aatggtggctgg tgggttcat attatgccctgt ccaccgggc actatt 1632 AsnGlyGlyTrp TrpValHis IleMetProCys ProProGly ThrIle tggtgtcaagaa aaattgact tgtataggcgaa 1665 TrpCysGlnGlu LysLeuThr CysIleGlyG1u <210> 18 <211> 555 <212> PRT

<213> Dermatophagoides farinae <400> 18 AL-2-C4-PCT.ST25.txt Met Lys Thr Ile Tyr Ala Ile Leu Ser Ile Met Ala Cys Ile Gly Leu Met Asn Ala Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met Arg Ile Val Cys Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val Asp Pro Tyr Thr Ile Glu Asp Ile Asp Pro Phe Lys Cys Thr His Leu Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val Phe Asp Pro Tyr Gln Asp Asp Asn His Asn Ser Trp Glu Lys Arg Gly Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Ile Gln Ser Val Leu Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Tle Asp Lys Gln Asn Tyr Leu Ala Leu Val Arg Glu Leu Lys Asp Ala Phe Glu Pro His Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly Lys Asp Lys Ile Asp Arg A1a Tyr Asp Ile Lys Glu Leu Asn Lys Leu Phe Asp Trp Met Asn Val Met Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn Phe Tyr Gly His Asn Ala Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr Tyr Phe AL-2-C4-PCT. ST25 . tact Asn Val Asn Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg Asp Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser Ile Glu Asp Arg Ser Lys Leu Lys Leu Gly Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Ser Gly Glu Glu Gly Val Leu Ser Tyr Ile Glu Leu Cys Gln Leu Phe Gln Lys Glu Glu Trp His Ile Gln Tyr Asp Glu Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr Asp Asp Leu Ala Ser Ile Ser Cys Lys Leu Ala Phe Leu Lys Glu Leu Gly Val Ser Gly Val Met Val Trp Ser Leu Glu Asn Asp Asp Phe Lys Gly His Cys Gly Pro Lys Asn Pro Leu Leu Asn Lys Val His Asn Met Ile Asn Gly Asp Glu Lys Asn Ser Phe Glu Cys Ile Leu Gly Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr Pro Thr Thr Pro Thr Thr Thr Pro Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr Ser Thr Pro Ser Pro Thr Thr Thr Glu His Thr Ser Glu Thr Pro Lys Tyr Thr Thr Tyr Val Asp Gly His Leu Ile Lys Cys Tyr Lys Glu Gly Asp AL-2-C4-PCT.ST25.txt Ile Pro His Pro Thr Asn Ile His Lys Tyr Leu Val Cys Glu Phe Val Asn Gly Gly Trp Trp Val His Ile Met Pro Cys Pro Pro Gly Thr Ile Trp Cys Gln Glu Lys Leu Thr Cys Ile Gly Glu <210>

<211>

<212>
DNA

<213>
Dermatophagoides farinae <400>

ttcgcctatacaagtcaatttttcttgacaccaaatagtgcccggtggacagggcataat60 atgaacccaccagccaccattaacaaattcacagaccaaatatttgtgtatattggttgg120 atgtgggatatcaccttccttgtaacatttgataagatgtccatcgacataggttgtata180 ttttggtgtttcgcttgtgtgttcggtcgtggttggcgaaggtgtcgatgttgtaggggC240 tggtgttggtgttgttggtgttggtgtggtgggagaaggagttgttgtcggtgtggtggg300 agaaggggttgttgtcggggtggtgggagaaggggttgttgtcggggtggtgggagaagg360 agttgttggcgttgtagtcggggttgtgggtgtcgtcgttggagttggtgtcgttgtact420 tggacccaaaatgcattcgaaagagttcttttcatcgccattaatcatattatgaacttt480 gttcaacaatggatttttcggtccgcagtgacctttgaaatcatcattttccaatgacca540 aaccatgacaccagaaacgcctaattctttcaggaaagccaacttgcatgatatactggc600 cagatcatcgtaaccgacccagattttatCattgtaaccatatggagcattgtaatattc660 atcgtattggatatgccattcttctttttgaaacaattgacacaattctatatatgagag720 gacaccttcttcaccagaaatgaaacctgggggcgacatgcctttggctggatctccaag780 tttgagtttgcttcgatcttcaatgctccaagcacggccatagaatggaacacccattac.840 caatttgtctctggtggcaccattgttcaaataatagtgcatggtgtagttgacattgaa900 gtaagtgtgcaactcatcagtttcatctggtcgtttatacaacggagcattgtgaccgta960 aaagttttcccatccaccgtggtaatcatatgtcatgacattcatccaatcgaacaattt1020 gttcaattctttgatatcataagctcggtcgattttgtctttacctggtgatactgcagc1080 agtcaacaagtagccatgaggttcaaaagcgtctttaagttctctaaccaaagccaaata1140 gttttgtttatcgattttcgggttacccaatcgagatccaggatactcccaatccaaatc1200 tagaccgtcgaacttgtattcttgcaaaaagtccaaaactgattgtatgaattgttgacg1260 atatgttggatttgcagccatatcggaatatttttccgagccttcataccaaccaccaag1320 tgaaatcatggtggttaattctggattcttcaatcgcaagttgttgaaacgttcataacc1380 AL-2-C4-PCT.ST25.txt acgtttttcccatgagttatggttatcatcttggtaaggatcgaaaacttgaattgtgta1440 tttgtattcatcaattttagcgaaaccatacattaaatgtgtacacttgaatggatcaat1500 atcttcgatagtgtatggatcaactttatgatatacggaccatgttccaacataacaaac1560 aattctcatcggatttttcgaataatcattatgatctcgtttgatggatgcattcataag1620 gccaatgcaggccataatactaagtattgcatatatggttttcat 1665 <210> 20 <211> 1608 <212> DNA
<213> Dermatophagoides farinae <220>
<221> CDS
<222> (1)..(1608) <223>
<400>

tcc atc,aaacga gatcataat gattattcg aaaaatccg atgagaatt 48 Ser IleLysArg AspHisAsn AspTyrSer LysAsnPro MetArgIle gtt tgttatgtt ggaacatgg tccgtatat cataaagtt gatccatac 96 Val CysTyrVal,GlyThrTrp SerValTyr HisLysVal AspProTyr act atcgaagat attgatcca ttcaagtgt acacattta atgtatggt 144 Thr IleGluAsp IleAspPro PheLysCys ThrHisLeu MetTyrGly 35 ~ 40 45 ttc getaaaatt gatgaatac aaatacaca attcaagtt ttcgatcct 192 Phe AlaLysIle AspGluTyr LysTyrThr IleGlnVal PheAspPro tac caagatgat aaccataac tcatgggaa aaacgtggt tatgaacgt 240 Tyr GlnAspAsp AsnHisAsn SerTrpGlu LysArgGly TyrGluArg ttc aacaacttg cgattgaag aatccagaa ttaaccacc atgatttca 288 Phe AsnAsnLeu ArgLeuLys AsnProGlu LeuThrThr MetIleSer ctt ggtggttgg tatgaaggc tcggaaaaa tattccgat atggetgca 336 Leu GlyGlyTrp TyrGluGly SerGluLys TyrSerAsp MetAlaAla aat ccaacatat cgtcaacaa ttcatacaa tcagttttg gactttttg 384 Asn ProThrTyr ArgGlnGln PheIleGln SerValLeu AspPheLeu caa gaatacaag ttcgacggt ctagatttg gattgggag tatcctgga 432 Gln GluTyrLys PheAspGly LeuAspLeu AspTrpGlu TyrProGly tct cgattgggt aacccgaaa atcgataaa caaaactat ttggetttg 480 Ser ArgLeuGly AsnProLys IleAspLys GlnAsnTyr LeuAlaLeu AL-2-C4-PCT.ST25.txt gttagagaactt aaagacget tttgaacct catggctac ttgttgact 528 ValArgGluLeu LysAspAla PheGluPro .HisGlyTyr LeuLeuThr getgcagtatca ccaggtaaa gacaaaatc gaccgaget tatgatatc 576 AlaAlaValSer ProGlyLys AspLysIle AspArgAla TyrAspIle aaagaattgaac aaattgttc gattggatg aatgtcatg acatatgat 624 LysGluLeuAsn LysLeuPhe AspTrpMet AsnValMet ThrTyrAsp taccacggtgga tgggaaaac ttttacggt cacaatget ccgttgtat 672 TyrHisGlyGly TrpGluAsn PheTyrGly HisAsnAla ProLeuTyr aaacgaccagat gaaactgat gagttgcac acttacttc aatgtcaac 720 LysArgProAsp GluThrAsp GluLeuHis ThrTyrPhe AsnValAsn tacaccatgcac tattatttg aacaatggt gccaccaga gacaaattg 768 TyrThrMetHis TyrTyrLeu AsnAsnGly AlaThrArg AspLysLeu gtaatgggtgtt Ccattctat ggccgtget tggagcatt gaagatcga 816 ValMetGlyVal ProPheTyr GlyArgAla TrpSerIle GluAspArg agcaaactcaaa cttggagat ccagccaaa ggcatgtcg cccccaggt 864 SerLysLeuLys LeuGlyAsp ProAlaLys GlyMetSer ProProGly ttcatttctggt gaagaaggt gtcctctca tatatagaa ttgtgtcaa 912 PheIleSerGly G1uGluGly ValLeuSer TyrIleGlu LeuCysGln ttgtttcaaaaa gaagaatgg catatccaa tacgatgaa tattacaat 960 LeuPheGlnLys GluGluTrp HisIleGln TyrAspGlu TyrTyrAsn getccatatggt tacaatgat aaaatctgg gtcggttac gatgatctg 1008 AlaProTyrGly TyrAsnAsp LysIleTrp ValGlyTyr AspAspLeu gccagtatatca tgcaagttg getttcctg aaagaatta ggcgtttct 1056 AlaSerIleSer CysLysLeu AlaPheLeu LysGluLeu GlyValSer ggtgtcatggtt tggtcattg gaaaatgat gatttcaaa ggtcaCtgc 1104 GlyValMetVal TrpSerLeu GluAsnAsp AspPheLys GlyHisCys ggaccgaaaaat ccattgttg aacaaagtt cataatatg attaatggc 1152 GlyProLysAsn ProLeuLeu AsnLysVa1 HisAsnMet IleAsnGly gatgaaaagaac tctttcgaa tgcattttg ggtccaagt acaacgaca 1200 AspGluLysAsn SerPheGlu CysIleLeu GlyProSer ThrThrThr ccaactccaacg acgacaccc acaaccccg actacaacg ccaacaact 1248 ProThrProThr ThrThrPro ThrThrPro ThrThrThr ProThrThr AL-2-C4-PCT.ST25.txt cct tctCCC aCCaCCCCgaca acaacc ccttctcccacc accccgaca 1296 Pro SerPro ThrThrProThr ThrThr ProSerProThr ThrProThr aca acccct tctcccaccaca ccgaca acaactccttct cccaccaca 1344 Thr ThrPro SerProThrThr ProThr ThrThrProSer ProThrThr cca acacca acaacaccaaca ccagcc cctacaacatcg acaccttcg 1392 Pro ThrPro ThrThrProThr ProAla ProThrThrSer ThrProSer cca accacg accgaacacaca agcgaa acaccaaaatat acaacctat 1440 Pro ThrThr ThrGluHisThr SerGlu ThrProLysTyr ThrThrTyr gtc gatgga catcttatcaaa tgttac aaggaaggtgat atcccacat 1488 Val AspGly HisLeuIleLys CysTyr LysGluGlyAsp IleProHis cca accaat atacacaaatat ttggtc tgtgaatttgtt aatggtggc 1536 Pro ThrAsn IleHisLysTyr LeuVal CysGluPheVal AsnGlyGly tgg tgggtt catattatgccc tgtcca ccgggcactatt tggtgtcaa 1584 Trp TrpVal HisI1eMetPro CysPro ProGlyThrIle TrpCysGln gaa aaattg acttgtataggc gaa 1608 Glu LysLeu ThrCysIleGly Glu <210> 21 <211> 53 <212> PRT

<213> Dermatophagoides farinae <400> 21 Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met Arg I1e Val Cys Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val Asp Pro Tyr Thr Ile Glu Asp Ile Asp Pro Phe Lys Cys Thr His Leu Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val Phe Asp Pro Tyr Gln Asp Asp Asn His Asn Ser Trp Glu Lys Arg Gly Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr Met Ile Ser AL-2-C4-PCT.ST25.txt Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Ile Gln Ser Va1 Leu Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln Asn Tyr Leu Ala Leu Val Arg Glu Leu Lys Asp A1a Phe Glu Pro His Gly Tyr Leu Leu Thr AlaAla Val Ser Pro Gly Lys Asp Lys Ile Asp Arg Ala Tyr Asp Tle Lys Glu Leu Asn Lys Leu Phe Asp Trp Met Asn Val Met Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn Phe Tyr Gly His Asn Ala Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr Tyr Phe Asn Val Asn Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala.Thr Arg Asp Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser Ile Glu Asp Arg Ser Lys Leu Lys Leu Gly Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Ser Gly Glu Glu Gly Val Leu Ser Tyr Ile Glu Leu Cys Gln Leu Phe Gln Lys Glu Glu Trp His Ile Gln Tyr Asp Glu Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr Asp Asp Leu Ala Ser Ile Ser Cys Lys Leu Ala Phe Leu Lys Glu Leu Gly Val Ser AL-2-C4-PCT.ST25.txt Gly Val Met Val Trp Ser Leu Glu Asn Asp Asp Phe Lys Gly His Cys Gly Pro Lys Asn Pro Leu Leu Asn Lys Val His Asn Met Ile Asn Gly Asp Glu Lys Asn Ser Phe Glu Cys Ile Leu Gly Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr Pro Thr Thr Pro Thr Thr Thr Pro Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr Pro Thr Thr Thr Pro Ser Pro Thr Thr 435 440 ~ 445 Pro Thr Pro Thr Thr Pro Thr Pro Ala Pro Thr Thr Ser Thr Pro Ser Pro Thr Thr Thr Glu His Thr Ser Glu Thr Pro Lys Tyr Thr Thr Tyr Val Asp G1y His Leu Ile Lys Cys Tyr Lys Glu Gly Asp I1e Pro His Pro Thr Asn Ile His Lys Tyr Leu Va1 Cys Glu Phe Val Asn Gly Gly Trp Trp Val His Ile Met Pro Cys Pro Pro Gly Thr Ile Trp Cys Gln Glu Lys Leu Thr Cys Ile Gly Glu <210>

<211>

<212>
DNA

<213>
Dermatophagoides farinae <400>

ttcgcctatacaagtcaatttttcttgacaccaaatagtgcccggtggacagggcataat60 atgaacccaccagccaccattaacaaattcacagaccaaatatttgtgtatattggttgg120 atgtgggatatcaccttccttgtaacatttgataagatgtccatcgacataggttgtata180 ttttggtgtttcgcttgtgtgttcggtcgtggttggcgaaggtgtcgatgttgtaggggc240 tggtgttggtgttgttggtgttggtgtggtgggagaaggagttgttgtcggtgtggtggg300 AL-2-C4-PCT.ST25.txt agaaggggttgttgtcggggtggtgggagaaggggttgttgtcggggtggtgggagaagg360 agttgttggcgttgtagtcggggttgtgggtgtcgtcgttggagttggtgtcgttgtact420 tggacccaaaatgcattcgaaagagttcttttcatcgccattaatcatattatgaacttt480 gttcaacaatggatttttcggtccgcagtgacctttgaaatcatcattttccaatgacca540 aaccatgacaccagaaacgcctaattctttcaggaaagccaacttgcatgatatactggc600 cagatcatcgtaaccgacccagattttatcattgtaaccatatggagcattgtaatattc660 atcgtattggatatgccattcttctttttgaaacaattgacacaattctatatatgagag720 gacaccttcttcaccagaaatgaaacctgggggcgacatgcctttggctggatctccaag780 tttgagtttgcttcgatcttcaatgctccaagcacggccatagaatggaacacccattac840 caatttgtctctggtggcaccattgttcaaataatagtgcatggtgtagttgacattgaa900 gtaagtgtgcaactcatcagtttcatctggtcgtttatacaacggagcattgtgaccgta960 aaagttttcccatccaccgtggtaatcatatgtcatgacattcatccaatcgaacaattt1020 gttcaattctttgatatcataagctcggtcgattttgtctttacctggtgatactgcagc1080 agtcaacaagtagccatgaggttcaaaagcgtctttaagttctctaaccaaagccaaata1140 gttttgtttatcgattttcgggttacccaatcgagatccaggatactcccaatccaaatc1200 tagaccgtcgaacttgtattcttgcaaaaagtccaaaactgattgtatgaattgttgacg1260 atatgttggatttgcagccatatcggaatatttttccgagccttcataccaaccaccaag1320 tgaaatcatggtggttaattctggattcttcaatcgcaagttgttgaaacgttcataacc1380 acgtttttcccatgagttatggttatcatcttggtaaggatcgaaaacttgaattgtgta1440 tttgtattcatcaattttagcgaaaccatacattaaatgtgtacacttgaatggatcaat,1500 atcttcgatagtgtatggatcaactttatgatatacggaccatgttccaacataacaaac1560 aattctcatcggatttttcgaataatcattatgatctcgtttgatgga 1608 <210> 23 <211> 25 <212> PRT
<213> Dermatophagoides farinae <220>
<221> MISC_FEATURE
<222> (1) . (1) <223> Xaa --- any amino acid at position 1 <400> 23 Xaa Leu Glu Pro Lys Thr Val Cys Tyr Tyr Glu Ser Trp Val His His Arg Gln Gly Glu Gly Lys Met Asp Pro AL-2-C4-PCT.ST25.txt <210> 24 <211> 33 <212> PRT
<213> Dermatophagoides farinae <220>
<221> MISC_FEATURE
<222> (18) .(18) <223> Xaa = any amino acid at position 18 <220>
<221> MISC_FEATURE
<222> (28) .(28) <223> Xaa = any amino acid at position 28 <220>
<221> MISC_FEATURE
<222> (31) . (31) <223> Xaa = any amino acid at position 31 <220>
<221> MISC_FEATURE
<222> (32) .(32) <223> Xaa = any amino acid at position 32 <400> 24 Ser Ile Lys Arg Asp His Asn Asp Tyr Ser Lys Asn Pro Met Met Ile Val Xaa Tyr Gly Gly Ser Ser Gly Tyr Gln Ser Xaa Lys Arg Xaa Xaa Thr <210> 25 <211> 31 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic Primer <220>
<221> misc_feature <222> (24) .(24) <223> n = a, c, t or g at position 24 <400> 25 aaacgtgatc ataaygatta ytcnaaraay c 31 <210> 26 <211> 31 <212> DNA
<213> Artificial sequence AL-2-C4-PCT.ST25.txt <220>
<223> Synthetic Primer <400> ~ 26 aaacgtgatc ataaygatta yagyaaraay c 31 <210> 27 <211> 23 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic Primer <220>
<221> misc_feature <222> (12) .(12) <223> n = a, c, t or g at position 12 <220>
<221> misc_feature <222> (21) .(21) <223> n = a, c, t or g at position 21 <400> 27 ccttcttcac cnacratcaa ncc 23 <210> 28 <211> 23 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic Primer <220>
<221> misc_feature <222> (12) .(12) <223> n = a, c, t or g at position 12 <220>
<221> misc-feature <222> (21) . . (21) <223> n = a, c, t or g at position 21 <400> 28 ccttcttcac cnacratgaa ncc 23 <210> 29 <211> 13 <212> PRT
<213> Dermatophagoides farinae AL-2-C4-PCT.ST25.txt <400> 29 Gln Tyr Gly Val Thr Gln Ala Val Val Thr Gln Pro Ala <210> 30 <211> 11 <212> PRT
<213> Dermatophagoides farinae <400> 30 Asp Glu Leu Leu Met Lys Ser Gly Pro Gly Pro <210> 31 <211> 24 <212> PRT
<213> Dermatophagoides farinae <400> 31 Asp Met Glu His Phe Thr Gln His Lys Gly Asn Ala Lys A1a Met Ile Ala Val Gly Gly Ser Thr Met Ser <210> 32 <211> 21 <212> PRT
<213> Dermatophagoides farinae <400> 32 Asp Ala Asn Glu Glu Ala Arg Ser Gln Leu Pro Glu Thr Ala Met Val Leu Ile Ly's Ser Gln <210> 33 <211> 21 <212> PRT
<213> Dermatophagoides farinae <220>
<221> MISC_FEATURE
<222> (11) .(11) <223> Xaa = any amino acid at position 11 <400> 33 Gln Ser Arg Asp Arg Asn Asp Lys Pro Tyr Xaa I1e Val Lys Lys Lys Lys Lys Ala Leu Asp AL-2-C4-PCT.ST25.txt <210> 34 <211> 1621 <212> DNA
<213> Dermatophagoides farinae <220>
<221> CDS
<222> (14)..(1540) <223>
<400>

agaacttatg tt 49 aaa gca atg ttg aaa ttt acg tgt aca ata t tgg gcc Met Lys Thr Thr Phe Ala Leu Phe Cys Ile Trp Ala tgcatt ggcttgatg aatgcg gccactaaa cgagatcac aataattat 97 CysIle GlyLeuMet AsnAla AlaThrLys ArgAspHis AsnAsnTyr tcgaaa aatccaatg cgaatc gtatgttat gttggaaca tggtccgtt 145 SerLys AsnProMet ArgIle ValCysTyr ValGlyThr TrpSerVal tatcat aaagttgat ccatac acaattgaa gatattgat cctttcaaa 193 TyrHis LysValAsp ProTyr ThrIleGlu AspIleAsp ProPheLys tgtact catttgatg tatggt tttgetaaa atcgatgaa tacaaatac 241 CysThr HisLeuMet TyrGly PheAlaLys TleAspGlu TyrLysTyr accatt caagttttt gatcca tttcaagat gataaccat aactcatgg 289 ThrIle GlnValPhe AspPro PheGlnAsp AspAsnHis AsnSerTrp gaaaaa cacgggtat gaacgt ttcaacaac ttgagattg aagaatcca 337 GluLys HisGlyTyr GluArg PheAsnAsn LeuArgLeu LysAsnPro gaattg accaccatg atttca ttgggtggt tggtatgaa ggttcagaa 385 GluLeu ThrThrMet 21eSer LeuGlyGly TrpTyrGlu GlySerGlu aaatat tcggatatg gcagcc aatccaaca tatcgtcag caatttgtt 433 LysTyr SerAspMet AlaAla AsnProThr TyrArgGln GlnPheVal Caatca gttttggac tttttg caagaatac aaattcgat ggcctagat 481 GlnSer ValLeuAsp PheLeu GlnGluTyr LysPheAsp GlyLeuAsp ttggat tgggaatat cctgga tcacggtta ggcaatcct aaaatcgat 529 LeuAsp TrpGluTyr ProGly SerArgLeu GlyAsnPro LysIleAsp aaacaa aactattta acatta gttagagaa cttaaagag gcatttgaa 577 LysGln AsnTyrLeu ThrLeu ValArgGlu LeuLysGlu AlaPheGlu AL-2-C4-PCT.ST25.txt cctttcggctac ttgttgact gccgcagta tcacccggtaaa gataaa 625 ProPheGlyTyr LeuLeuThr AlaAlaVal SerProG1yLys AspLys attgacgtaget tatgagctc aaagaattg aaccaattgttc gattgg 673 IleAspValAla TyrGluLeu LysGluLeu AsnGlnLeuPhe AspTrp atgaatgtcatg acttatgat taccatggc ggatgggaaaat gttttc 721 MetAsnValMet ThrTyrAsp TyrHisGly GlyTrpGluAsn ValPhe ggccataatget ccgttgtat aaacgaccc gatgaaacggat gaattg 769 G1yHisAsnAla ProLeuTyr LysArgPro AspGluThrAsp GluLeu cacacttacttc aatgtcaac tacaccatg cactattatttg aacaat 817 HisThrTyrPhe AsnValAsn TyrThrMet HisTyrTyrLeu AsnAsn ggcgetactcga gacaaactt gttatgggt gttccattctat ggtcgt 865 GlyAlaThr.Arg AspLysLeu ValMetGly ValProPheTyr GlyArg gettggagcatc gaagatcga agcaaagtc.aaacttggcgat CCggCC 913 AlaTrpSerIle GluAspArg SerLysVal LysLeuGlyAsp ProAla aaaggcatgtct cctcctggt tttattact ggtgaagaaggt gttctc 961 LysGlyMetSer ProProGly PheIleThr GlyGluGluGly Va1Leu tcatacatcgaa ttgtgtcag ttattccag aaagaagaatgg catatt 1009 SerTyrIleGlu LeuCysGln LeuPheGln LysGluGluTrp HisIle, caatacgatgaa tattacaat getccatac ggatataatgat aaaatc 1057 GlnTyrAspGlu TyrTyrAsn AlaProTyr GlyTyrAsnAsp LysIle tgggt~ ggttac gatgatctg getagtata tcatgcaagttg gccttt 1105 TrpVal GlyTyr AspAspLeu AlaSerIle SerCysLysLeu AlaPhe ctcaaa gaattg ggcgtctct ggcgttatg atatggtcattg gaaaac 1153 LeuLys GluLeu GlyValSer GlyValMet IleTrpSerLeu GluAsn gatgat ttcaaa ggtcattgc ggaccgaaa tatccattgttg aacaaa 1201 AspAsp PheLys GlyHisCys GlyProLys TyrProLeuLeu AsnLys gttcac aatatg atcaatggt gatgaaaag aactcttacgaa tgtctt 1249 ValHis AsnMet IleAsnGly AspGluLys AsnSerTyrGlu CysLeu ttgggc ccaagt acaaccaca ccaacacca accaccccgtca actact 1297 LeuGly ProSer ThrThrThr ProThrPro ThrThrProSer ThrThr tcgact accaca cca.acgcct accaccacc gatagcacaagc gaaaca 1345 SerThr ThrThr ProThrPro ThrThrThr AspSerThrSer GluThr AL-2-C4-PCT.ST25.txt ccaaaatacactacg tatattgat ggacatttg attaaatgc tataaa 1393 ProLysTyrThrThr TyrIleAsp GlyHisLeu IleLysCys TyrLys caaggttatcttcca catccaact gatgttcat aaatattta gtttgt 1441 GlnGlyTyrLeuPro HisProThr AspValHis LysTyrLeu ValCys gaatatattgccaca ccaaacggt ggttggtgg gtacacatt atggat 1489 GluTyrIleAlaThr ProAsnGly GlyTrpTrp ValHisIle MetAsp tgtccaaaaggaact agatggcac gcaacatta aaaaattgt attcaa 1537 CysProLysGlyThr ArgTrpHis AlaThrLeu LysAsnCys IleGln gaatgatctgata tatttgtaac taaatgaaat ttaaataaaa tgttttttgc Glu ttatttgaat ccattaaaaa aaaaaaaaaa a 1621 <210> 35 <211> 509 <212> PRT
<213> Dermatophagoides farinae <400> 35 Met Lys Thr Thr Phe Ala Leu Phe Cys Ile Trp Ala Cys Ile Gly Leu Met Asn Ala Ala Thr Lys Arg Asp His Asn Asn Tyr Ser Lys Asn Pro Met Arg Ile Val Cys Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val Asp Pro Tyr Thr Ile Glu Asp Ile Asp Pro Phe Lys Cys Thr His Leu Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val 65 70, 75 80 Phe Asp Pro Phe Gln Asp Asp Asn His Asn Ser Trp Glu Lys His G1y Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Val Gln Ser Val Leu 130 ' 135 140 AL-2-C4-PCT.ST25.txt Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln Asn Tyr Leu Thr Leu Val Arg Glu Leu Lys G1u Ala Phe Glu Pro Phe Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly Lys Asp Lys Ile Asp Val Ala Tyr G1u Leu Lys Glu Leu Asn Gln Leu Phe Asp Trp Met Asn Val Met Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn Val Phe Gly His Asn Ala 225 '230 235 240 Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr Tyr Phe Asn Va1 Asn Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg Asp Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser Ile Glu Asp Arg Ser Lys Val Lys Leu Gly Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Thr Gly Glu Glu Gly Val Leu Ser Tyr Ile Glu Leu Cys Gln Leu Phe Gln Lys Glu Glu Trp His Ile Gln Tyr Asp Glu Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr Asp Asp Leu Ala Ser Ile Ser Cys Lys Leu Ala Phe Leu Lys Glu Leu Gly Val Ser Gly Val Met Ile Trp Ser Leu Glu Asn Asp Asp Phe Lys Gly His Cys Gly Pro Lys Tyr Pro Leu Leu Asn Lys Val His Asn Met AL-2-C4-PCT.ST25.txt Ile Asn Gly Asp Glu Lys Asn Ser Tyr Glu Cys Leu Leu Gly Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr Pro Ser Thr Thr Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr Asp Ser Thr Ser Glu Thr Pro Lys Tyr Thr Thr Tyr Ile Asp Gly His Leu Ile Lys Cys Tyr Lys Gln Gly Tyr Leu Pro His Pro Thr Asp Val His Lys Tyr Leu Val Cys Glu Tyr Ile Ala Thr Pro Asn Gly Gly Trp Trp Val His Ile Met Asp Cys Pro Lys Gly Thr Arg Trp His Ala Thr Leu Lys Asn Cys Ile Gln Glu <210> 36 <211> 1621 <212> DNA
<213> Dermatophagoides farinae <400> 36 tttttttttt ttttttaatg gattcaaata attttattta aatttcattt agcaaaaaac 60 agttacaaatatatcagatcattcttgaatacaattttttaatgttgcgtgccatctagt120 tccttttggaCaatccataatgtgtacccaccaaccaccgtttggtgtggcaatatattc180 acaaactaaatatttatgaacatcagttggatgtggaagataaccttgtttatagcattt240 aatcaaatgtccatcaatatacgtagtgtattttggtgtttcgcttgtgctatcggtggt300 ggtaggcgttggtgtggtagtcgaagtagttgacggggtggttggtgttggtgtggttgt360 acttgggcccaaaagacattcgtaagagttcttttcatcaCcattgatcatattgtgaac420 tttgttcaacaatggatatttcggtccgcaatgacctttgaaatcatcgttttccaatga480 ccatatcataacgccagagacgcccaattctttgagaaaggccaacttgcatgatatact540 agccagatcatcgtaaccaacccagattttatcattatatccgtatggagcattgtaata600 ttcatcgtattgaatatgccattcttctttctggaataactgacacaattcgatgtatga660 gagaacaccttcttcaccagtaataaaaccaggaggagacatgcctttggccggatcgcc720 aagtttgactttgcttcgatcttcgatgctccaagcacgaccatagaatggaacacccat780 aacaagtttgtctcgagtagcgccattgttcaaataatagtgcatggtgtagttgacatt840 AL-2-C4-PCT.ST25.txt gaagtaagtgtgcaattcatccgtttcatcgggtcgtttatacaacggagcattatggcc900 gaaaacattttcccatccgccatggtaatcataagtcatgacattcatccaatcgaacaa960 ttggttcaattctttgagctcataagctacgtcaattttatctttaccgggtgatactgc1020 ggcagtcaacaagtagccgaaaggttcaaatgcctctttaagttctctaactaatgttaa1080 atagttttgtttatcgattttaggattgcctaaccgtgatccaggatattcccaatccaa1140 atctaggccatcgaatttgtattcttgcaaaaagtccaaaactgattgaacaaattgctg1200 acgatatgttggattggctgccatatccgaatatttttctgaaccttcataccaaccacc1260 caatgaaatcatggtggtcaattctggattcttcaatctcaagttgttgaaacgttcata1320 cccgtgtttttcccatgagttatggttatcatcttgaaatggatcaaaaacttgaatggt1380 gtatttgtattcatcgattttagcaaaaccatacatcaaatgagtacatttgaaaggatc1440 aatatcttcaattgtgtatggatcaactttatgataaacggaccatgttccaacataaca1500 tacgattcgcattggatttttcgaataattattgtgatctcgtttagtggccgcattcat1560 caagccaatgcaggcccatatacaaaacaatgcaaatgtcgttttcattttcataagttc1620 t 1621 <210>

<211>

<212>
DNA

<213>
Dermatophagoides farinae <220>
<221> CDS
<222> (1)..(1527) <223>
<400>

atgaaa acgaca tttgcattg ttttgtata tgggcctgcatt ggcttg 48 MetLys ThrThr PheAlaLeu PheCysIle TrpAlaCysIle GlyLeu atgaat gcggcc actaaacga gatcacaat aattattcgaaa aatcca 96 MetAsn AlaAla ThrLys'ArgAspHisAsn AsnTyrSerLys AsnPro atgcga atcgta tgttatgtt ggaacatgg-tccgtttatcat aaagtt 144 MetArg IleVal CysTyrVal GlyThrTrp SerValTyrHis LysVal gatcca tacaca attgaagat attgatcct ttcaaatgtact catttg 192 AspPro TyrThr IleGluAsp IleAspPro PheLysCysThr HisLeu atgtat ggtttt getaaaatc gatgaatac aaatacaccatt caagtt 240 MetTyr GlyPhe AlaLysIle AspGluTyr LysTyrThrIle GlnVal tttgat ccattt caagatgat aaccataac tcatgggaaaaa cacggg 288 PheAsp ProPhe GlnAspAsp AsnHisAsn SerTrpGluLys HisGly AL-2-C4-PCT.ST25.txt tatgaacgtttc aacaacttg agattgaagaat ccagaattg accacc 336 TyrGluArgPhe AsnAsnLeu ArgLeuLysAsn ProGluLeu ThrThr atgatttcattg ggtggttgg tatgaaggttca gaaaaatat tcggat 384 MetIleSerLeu GlyGlyTrp TyrGluGlySer GluLysTyr SerAsp atggcagccaat ccaacatat cgtcagcaattt gttcaatca gttttg 432 MetAlaAlaAsn ProThrTyr ArgGlnGlnPhe ValGlnSer ValLeu gactttttgcaa gaatacaaa ttcgatggccta gatttggat tgggaa 480 AspPheLeuGln GluTyrLys PheAspG1yLeu AspLeuAsp TrpGlu tatCctggatca cggttaggc aatcctaaaatc gataaacaa aactat 528 TyrProGlySer ArgLeuGly AsnProLysIle AspLysGln AsnTyr ttaacattagtt agagaactt aaagaggcattt gaacctttc ggctac 576 LeuThrLeuVal ArgGluLeu LysGluAlaPhe GluProPhe GlyTyr ttgttgactgcc gcagtatca cccggtaaagat aaaattgac gtaget 624 LeuLeuThrAla AlaValSer ProGlyLysAsp LysT1eAsp ValAla tatgagctcaaa gaattgaac caattgttcgat tggatgaat gtcatg 672 TyrGluLeuLys GluLeuAsn GlnLeuPheAsp TrpMetAsn ValMet acttatgattac catggcgga tgggaaaatgtt ttcggccat aatget 720 ThrTyrAspTyr HisGlyGly TrpGluAsnVal PheGlyHis AsnAla ccgttgtataaa cgacccgat gaaacggatgaa ttgcacact taCttc 768 ProLeuTyrLys ArgProAsp GluThrAspGlu LeuHisThr TyrPhe aatgtcaactac accatgcac tattatttgaac aatggcget actcga 816 AsnValAsnTyr ThrMetHis TyrTyrLeuAsn AsnGlyAla ThrArg gacaaacttgtt atgggtgtt ccattctatggt cgtgettgg agcatc 864 AspLysLeuVal MetGlyVal ProPheTyrGly ArgAlaTrp SerIle gaagatcgaagc aaagtcaaa cttggcgatccg gccaaaggc atgtct 912 GluAspArgSer LysValLys LeuGlyAspPro AlaLysGly MetSer cctcctggtttt attactggt gaagaaggtgtt ctctcatac atcgaa 960 ProProGlyPhe IleThrGly GluGluGlyVal LeuSerTyr IleGlu ttgtgtcagtta ttccagaaa gaagaatggcat attcaatac gatgaa 1008 LeuCysGlnLeu PheGlnLys GluGluTrpHis IleGlnTyr AspGlu tattacaatget ccatacgga tataatgataaa atctgggtt ggttac 1056 TyrTyrAsnAla ProTyrGly TyrAsnAspLys IleTrpVal GlyTyr AL-2-C4-PCT.ST25.txt gatgat ctgget agtatatca tgcaagttggcc tttctcaaa gaattg 1104 AspAsp LeuAla SerIleSer CysLysLeuAla PheLeuLys GluLeu ggcgtc tctggc gttatgata tggtcattggaa aacgatgat ttcaaa 1152 GlyVal SerGly ValMetI1e TrpSerLeuGlu AsnAspAsp PheLys ggtcat tgcgga ccgaaatat ccattgttgaac aaagttcac aatatg 1200 GlyHis CysGly ProLysTyr ProLeuLeuAsn LysValHis AsnMet atcaat ggtgat gaaaagaac tcttacgaatgt Cttttgggc ccaagt 1248.

IleAsn GlyAsp GluLys.AsnSerTyrGluCys LeuLeuGly ProSer acaacc acacca acaccaacc accccgtcaact acttcgact accaca 1296 ThrThr ThrPro ThrProThr ThrProSerThr ThrSerThr ThrThr Ccaacg cctacc accaccgat agcacaagcgaa acaccaaaa tacact 1344 ProThr ProThr ThrThrAsp SerThrSerGlu ThrProLys TyrThr 435 440 445.

acgtatattgat ggacatttg attaaatgc tataaacaaggt tatctt 1392 ThrTyrIleAsp G1yHisLeu IleLysCys TyrLysG1nGly TyrLeu ccacatccaact gatgttcat aaatattta gtttgtgaatat attgcc 1440 .

ProHisProThr AspValHis LysTyrLeu ValCysGluTyr IleAla acaccaaacggt ggttggtgg gtacacatt atggattgtcca aaagga 1488 ThrProAsnGly GlyTrpTrp ValHisIle MetAspCysPro LysGly actagatggcac gcaacatta aaaaattgt attcaagaa 1527 ThrArgTrpHis AlaThrLeu LysAsnCys IleGln.Glu <210> 38 <211> 509 <212> PRT
<213> Dermatophagoides farinae <400> 38 Met Lys Thr Thr Phe Ala Leu Phe Cys Ile Trp Ala Cys Ile Gly Leu Met Asn Ala Ala Thr Lys Arg Asp His Asn Asn Tyr Ser Lys Asn Pro Met Arg Ile Val Cys Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val Asp Pro Tyr Thr Tle Glu Asp Ile Asp Pro Phe Lys Cys Thr His Leu AL-2-C4-PCT.ST25.txt Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Ile Gln Val Phe Asp Pro Phe Gln Asp Asp Asn His Asn Ser Trp Glu Lys His Gly Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Val Gln Ser Val Leu Asp Phe Leu Gln Glu Tyr Lys Phe,Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln Asn Tyr Leu_Thr Leu Val Arg Glu Leu Lys Glu Ala Phe Glu Pro Phe Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly Lys Asp Lys 21e Asp Val Ala Tyr Glu Leu Lys Glu Leu Asn Gln Leu Phe Asp Trp Met Asn Val Met Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn Val Phe Gly His Asn Ala Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp G1u Leu His Thr Tyr Phe Asn Val Asn Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg Asp Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser Ile Glu Asp Arg Ser Lys Val Lys Leu Gly Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Thr Gly Glu Glu Gly Val Leu Ser Tyr Ile Glu AL-2-C4-PCT.ST25.txt Leu Cys Gln Leu Phe Gln Lys Glu Glu Trp His Ile Gln Tyr Asp G1u Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr Asp Asp Leu Ala Ser Ile Ser Cys Lys Leu Ala Phe Leu Lys Glu Leu Gly Val Ser Gly Val Met Ile Trp Ser Leu Glu Asn Asp Asp Phe Lys Gly His Cys Gly Pro Lys Tyr Pro Leu Leu Asn Lys Val His Asn Met Ile Asn Gly Asp Glu Lys Asn Ser Tyr Glu Cys Leu Leu Gly Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr Pro Ser Thr Thr Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr Asp Ser Thr Ser Glu Thr Pro Lys Tyr Thr Thr Tyr I1e Asp Gly His Leu Ile Lys Cys Tyr Lys Gln Gly Tyx Leu Pro His Pro Thr Asp Val His Lys Tyr Leu Val Cys Glu Tyr Ile Ala Thr Pro Asn Gly Gly Trp Trp Val His Ile Met Asp Cys Pro Lys Gly Thr Arg Trp His Ala Thr Leu Lys Asn Cys Ile Gln Glu <210>

<211>

<212>
DNA

<213>
Dermatophagoides farinae <400>

ttcttgaatacaattttttaatgttgcgtgccatctagttccttttggacaatccataat60 gtgtacccaccaaccaccgtttggtgtggcaatatattcacaaactaaatatttatgaac120 atcagttggatgtggaagataaccttgtttatagcatttaatcaaatgtccatcaatata180 cgtagtgtattttggtgtttcgcttgtgctatcggtggtggtaggcgttggtgtggtagt240 cgaagtagttgacggggtggttggtgttggtgtggttgtacttgggcccaaaagacattc300 AL-2-C4-PCT.ST25.txt gtaagagttcttttcatcaccattgatcatattgtgaactttgttcaacaatggatattt360 cggtccgcaatgacctttgaaatcatcgttttccaatgaccatatcataacgocagagac420 gcccaattctttgagaaaggccaacttgcatgatatactagccagatcatcgtaaccaac480 ccagattttatcattatatccgtatggagcattgtaatattcatcgtattgaatatgcca540 ttcttctttctggaataactgacacaattcgatgtatgagagaacaccttcttcaccagt600 aataaaaccaggaggagacatgcctttggccggatcgccaagtttgactttgcttcgatc660 ttcgatgctccaagcacgaccatagaatggaacacccataacaagtttgtctcgagtagc720 gccattgttcaaataatagtgcatggtgtagttgacattgaagtaagtgtgcaattcatc780 cgtttcatcgggtcgtttatacaacggagcattatggccgaaaacattttcccatccgcc840 atggtaatcataagtcatgacattcatccaatcgaacaattggttcaattctttgagctc900 ataagctacgtcaattttatctttaccgggtgatactgcggcagtcaacaagtagccgaa960 aggttcaaatgcctctttaagttctctaactaatgttaaatagttttgtttatcgatttt1020 aggattgcctaaccgtgatccaggatattcccaatccaaatctaggccatcgaatttgta1080 ttcttgcaaaaagtccaaaactgattgaacaaattgctgacgatatgttggattggctgc1140 catatccgaatatttttctgaaccttcataccaacCacccaatgaaatcatggtggtcaa1200 "

ttctggattcttcaatctcaagttgttgaaacgttcatacccgtgtttttcccatgagtt1260 atggttatcatcttgaaatggatcaaaaacttgaatggtgtatttgtattcatcgatttt1320 agcaaaaccatacatcaaatgagtacatttgaaaggatcaatatcttcaattgtgtatgg1380 atcaactttatgataaacggaccatgttccaacataacatacgattcgcattggattttt1440 cgaataattattgtgatctcgtttagtggccgcattcatcaagccaatgcaggcccatat1500 acaaaacaatgcaaatgtcgttttcat 1527 <210>40 <211>1470 <212>DNA

<213>Dermatophagoides farinae <220>

<221>CDS

<222>(1)..(1470).

<223>

<400> 40 gcc act aaa cga gat cac aat aat tat tcg aaa aat cca atg cga atc 48 A1a Thr Lys Arg Asp His Asn Asn Tyr Ser Lys Asn Pro Met Arg Ile gta tgt tat gtt gga aca tgg tcc gtt tat cat aaa gtt gat cca tac 96 Val Cys Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val Asp Pro Tyr AL-2-C4-PCT.ST25.txt acaattgaagat attgatcct ttcaaatgtact catttgatg tatggt 144 ThrIleGluAsp IleAspPro PheLysCysThr HisLeuMet TyrGly tttgetaaaatc gatgaatac aaatacaccatt caagttttt gatcca 192 PheAlaLysIle AspGluTyr LysTyrThrIle GlnValPhe AspPro tttcaagatgat aaccataac tcatgggaaaaa cacgggtat gaacgt 240 PheGlnAspAsp AsnHisAsn SerTrpGluLys HisGlyTyr GluArg ttcaacaacttg agattgaag aatccagaattg accaccatg atttca 288 PheAsnAsnLeu ArgLeuLys AsnProGluLeu ThrThrMet IleSer ttgggtggttgg tatgaaggt tcagaaaaatat tcggatatg gcagcc 336 LeuGlyGlyTrp TyrG1uGly SerGluLysTyr SerAspMet AlaAla aatccaacatat cgtcagcaa tttgttcaatca gttttggac tttttg 384 AsnProThrTyr ArgGlnGln PheValGlnSer ValLeuAsp PheLeu caagaatacaaa ttcgatggc ctagatttggat tgggaatat cctgga 432 GlnGluTyrLys PheAspGly LeuAspLeuAsp TrpGluTyr ProGly tcacggttaggc aatcctaaa atcgataaacaa aactattta acatta 480 SerArgLeuGly AsnProLys IleAspLysGln AsnTyrLeu ThrLeu gttagagaactt aaagaggca tttgaacctttc ggctacttg ttgact 528 ValArgGluLeu LysGluAla PheGluProPhe GlyTyrLeu LeuThr gccgcagtatca cccggtaaa gataaaattgac gtagettat gagctc 576 AlaAlaValSer ProGlyLys AspLysIleAsp ValAlaTyr GluLeu aaagaattgaac caattg.ttcgattggatgaat gtcatgact tatgat 624 LysGluLeuAsn GlnLeuPhe AspTrpMetAsn ValMetThr TyrAsp taccatggcgga tgggaaaat gttttcggccat aatgetccg ttgtat 672 TyrHisGlyGly TrpGluAsn ValPheGlyHis AsnAlaPro LeuTyr aaacgacccgat gaaacggat gaattgcacact tacttcaat gtcaac 720 LysArgProAsp GluThrAsp GluLeuHisThr TyrPheAsn ValAsn tacaccatgcac tattatttg aacaatggcget actcgagac aaactt 768 TyrThrMetHis TyrTyrLeu AsnAsnGlyAla ThrArgAsp LysLeu gttatgggtgtt ccattctat ggtcgtgettgg agcatcgaa gatcga 816 ValMetGlyVal ProPheTyr GlyArgAlaTrp Sex'IleGlu AspArg agcaaagtcaaa cttggcgat ccggccaaaggc atgtctcct cctggt 864 SerLysValLys LeuGlyAsp ProAlaLysGly MetSerPro ProGly AL-2-C4-PCT.ST25. txt ttt attactggt gaagaaggt gttctctca tacatcgaa ttgtgtcag 912 Phe IleThrGly GluGluGly ValLeuSer TyrIleGlu LeuCysGln tta ttccagaaa gaagaatgg catattcaa tacgatgaa tattacaat 960 Leu PheGlnLys GluGluTrp HisIleGln TyrAspGlu TyrTyrAsn get ccatacgga tataatgat aaaatctgg gttggttac gatgatctg 1008 Ala ProTyrGly TyrAsnAsp LysIleTrp ValGlyTyr AspAspLeu get agtatatca tgcaagttg gcctttctc aaagaattg ggcgtctct 1056 Ala SerI1eSer CysLysLeu AlaPheLeu LysGluLeu GlyVa1Ser ggc gttatgata tggtcattg gaaaacgat gatttcaaa ggtcattgc 1104 Gly ValMetIle TrpSerLeu GluAsnAsp AspPheLys GlyHisCys gga ccgaaatat ccattgttg aacaaagtt cacaatatg atcaatggt 1152 Gly ProLysTyr ProLeuLeu AsnLysVal HisA'snMet TleAsnGly gat gaaaagaac tcttacgaa tgtcttttg ggcccaagt acaaccaca 1200 Asp G1uLysAsn SerTyrGlu CysLeuLeu GlyProSer ThrThrThr cca acaccaacc accccgtca actacttcg actaccaca ccaacgcct 1248 Pro ThrProThr ThrProSer ThrThrSer ThrThrThr ProThrPro acc accaccgat agcacaagc gaaacacca aaatacact acgtatatt 1296 Thr ThrThrAsp SerThrSer GluThrPro LysTyrThr ThrTyrI1e gat ggacatttg attaaatgc tataaacaa ggttatctt ccacatcca 1344 Asp GlyHisLeu IleLysCys TyrLysGln GlyTyrLeu ProHisPro act gatgttcat aaatattta gtttgtgaa tatattgcc acaccaaac 1392 Thr AspValHis LysTyrLeu ValCysGlu TyrIleAla ThrProAsn ggt ggttggtgg gtacacatt atggattgt ccaaaagga actagatgg 1440 Gly GlyTrpTrp ValHisIle MetAspCys ProLysGly ThrArgTrp cac gcaacatta aaaaattgt attcaagaa 1470 His AlaThrLeu LysAsnCys 21eGlnGlu <210> 41 <211> 490 <212> PRT

<213> Dermatophagoides farinae <400> 41 Ala Thr Lys Arg Asp His Asn Asn Tyr Ser Lys Asn Pro Met Arg Ile AL-2-C4-PCT.ST25.txt Val Cys Tyr Val Gly Thr Trp Ser Val Tyr His Lys Val Asp Pro Tyr Thr Ile~Glu Asp Ile Asp Pro Phe Lys Cys Thr His Leu Met Tyr Gly Phe Ala Lys Ile Asp Glu Tyr Lys Tyr Thr Tle Gln Val Phe Asp Pro Phe Gln Asp Asp Asn His Asn Ser Trp Glu Lys His Gly Tyr Glu Arg Phe Asn Asn Leu Arg Leu Lys Asn Pro Glu Leu Thr Thr Met Ile Ser Leu Gly Gly Trp Tyr Glu Gly Ser Glu Lys Tyr Ser Asp Met Ala Ala Asn Pro Thr Tyr Arg Gln Gln Phe Val Gln Ser Val Leu Asp Phe Leu Gln Glu Tyr Lys Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Arg Leu Gly Asn Pro Lys Ile Asp Lys Gln Asn Tyr Leu Thr Leu Val Arg Glu Leu Lys Glu Ala Phe Glu Pro Phe Gly Tyr Leu Leu Thr Ala Ala Val Ser Pro Gly Lys Asp Lys Ile Asp Val Ala Tyr Glu Leu l80 185 1.90 Lys Glu Leu Asn Gln Leu Phe Asp Trp Met Asn Val Met Thr Tyr Asp Tyr His Gly Gly Trp Glu Asn Val Phe Gly His Asn Ala Pro Leu Tyr Lys Arg Pro Asp Glu Thr Asp Glu Leu His Thr Tyr Phe Asn Val Asn Tyr Thr Met His Tyr Tyr Leu Asn Asn Gly Ala Thr Arg Asp Lys Leu Val Met Gly Val Pro Phe Tyr Gly Arg Ala Trp Ser Ile Glu Asp Arg AL-2-C4-PCT.ST25.txt Ser Lys Val Lys Leu Gly Asp Pro Ala Lys Gly Met Ser Pro Pro Gly Phe Ile Thr Gly Glu Glu Gly Val Leu Ser Tyr Ile Glu Leu Cys Gln Leu Phe Gln Lys Glu Glu Trp His Ile Gln Tyr Asp Glu Tyr Tyr Asn Ala Pro Tyr Gly Tyr Asn Asp Lys Ile Trp Val Gly Tyr Asp Asp Leu Ala Ser Ile Ser Cys Lys Leu Ala Phe Leu Lys Glu Leu Gly Va1 Ser Gly Val Met Ile Trp Ser Leu Glu Asn Asp Asp Phe Lys Gly His Cys Gly Pro Lys Tyr Pro Leu Leu Asn Lys Val His Asn Met,Ile Asn Gly Asp Glu Lys Asn Ser Tyr Glu Cys Leu Leu Gly Pro Ser Thr Thr Thr Pro Thr Pro Thr Thr Pro Ser Thr Thr Ser Thr Thr Thr Pro Thr Pro Thr Thr Thr Asp Ser Thr Ser Glu Thr Pro Lys Tyr Thr Thr Tyr Ile Asp Gly His Leu Ile Lys Cys Tyr Lys Gln Gly Tyr Leu Pro His Pro Thr Asp Val His Lys Tyr Leu Val Cys Glu Tyr Ile Ala Thr Pro Asn Gly Gly Trp Trp Val His Ile Met Asp Cys Pro Lys G1y Thr Arg Trp His Ala Thr Leu Lys Asn Cys Ile Gln Glu 485 ' 490 <210> 42 <211> 1470 <212> DNA
<213> Dermatophagoides farinae <400> 42 ttcttgaata caatttttta atgttgcgtg ccatctagtt ccttttggac aatccataat 60 AL-2-C4-PCT.ST25.txt gtgtacccac caaccaccgtttggtgtggcaatatattcacaaactaaatatttatgaac120 atcagttgga tgtggaagataaccttgtttatagcatttaatcaaatgtccatcaatata180 cgtagtgtat tttggtgtttcgcttgtgctatcggtggtggtaggcgttggtgtggtagt240 cgaagtagtt gacggggtggttggtgttggtgtggttgtacttgggcccaaaagacattc300 gtaagagttc ttttcatcaccattgatcatattgtgaactttgttcaacaatggatattt360 cggtccgcaa tgacctttgaaatcatcgttttccaatgaccatatcataacgccagagac420 gcccaattct ttgagaaaggccaacttgcatgatatactagccagatcatcgtaaccaac480 ccagatttta tcattatatccgtatggagcattgtaatattcatcgtattgaatatgcca540 ttcttctttc tggaataactgacacaattcgatgtatgagagaacaccttcttcaccagt.600 aataaaacca ggaggagacatgcctttggccggatcgccaagtttgactttgcttcgatc660 ttcgatgctc caagcacgaccatagaatggaacacccataacaagtttgtctcgagtagc720 gccattgttc aaataatagtgcatggtgtagttgacattgaagtaagtgtgcaattcatc780 cgtttcatcg ggtcgtttatacaacggagcattatggccgaaaacattttcccatccgcc840 atggtaatca taagtcatgacattcatccaatcgaacaattggttcaattctttgagctc900 ataagctacg tcaattttatctttaccgggtgatactgcggcagtcaacaagtagccgaa960 aggttcaaat gcctctttaagttctctaactaatgttaaatagttttgtttatcgatttt1020 aggattgcct aaccgtgatccaggatattcccaatccaaatctaggccatcgaatttgta1080 ttcttgcaaa aagtccaaaactgattgaacaaattgctgacgatatgttggattggctgc1140 catatccgaa tatttttctgaaccttcataccaaccacccaatgaaatcatggtggtcaa1200 ttctggattc ttcaatctcaagttgttgaaacgttcatacccgtgtttttcccatgagtt1260 atggttatca tcttgaaatggatcaaaaacttgaatggtgtatttgtattcatcgatttt1320 agcaaaacca tacatcaaatgagtacatttgaaaggatcaatatcttcaattgtgtatgg1380 atcaacttta tgataaacggaccatgttccaacataacatacgattcgcattggattttt1440 cgaataatta ttgtgatctcgtttagtggc 1470 <210> 43 <211> 510 <212> DNA
<213> Dermatophagoides farinae <220>
<221> CDS
<222> (1)..(510) <223>
<400> 43 gat atg gaa cat ttt aca caa cat aag ggc aac gcc aaa gcc atg atc 48 Asp Met G1u His Phe Thr G1n His Lys Gly Asn Ala Lys Ala Met Ile AL-2-C4-PCT.ST25.txt gccgtcggtggt tcgactatg tccgatcaattt tccaagact gcagcg 96 AlaValGlyGly SerThrMet SerAspGlnPhe SerLysThr AlaAla gtagaacattat cgggaaacg tttgttgttagc acagttgat cttatg 144 ValGluHisTyr ArgGluThr PheValValSer ThrValAsp LeuMet actcgttatggt ttcgatggt gtcatgattgat tggtctggc atgcaa 192 ThrArgTyrGly PheAspGly ValMetIleAsp TrpSerGly MetGln gccaaagatagt gataatttc attaaattgttg gacaaattc gacgaa 240 AlaLysAspSer AspAsnPhe IleLysLeuLeu AspLysPhe AspGlu aagtttgetcac acctcgttt gtgatgggtgtt accttgccg gcaacg 288 LysPheA1aHis ThrSerPhe ValMetGlyVal ThrLeuPro AlaThr atcgcatcatac gataactat aacattcctgcc atctccaac tatgtc 336 IleAlaSerTyr AspAsnTyr AsnIleProAla IleSerAsn TyrVal gattttatgaac gtgcttagt ctggattacact ggatcatgg gcccat 384 AspPheMetAsn ValLeuSer LeuAspTyrThr GlySerTrp AlaHis acggtcggtcat gettctccg tttcctgaacaa ctcaaaacg ctagaa 432 ThrValGlyHis AlaSerPro PheProGluGln LeuLysThr LeuGlu gettaccacaaa cgaggcget ccacgtcataag atggtcatg getgta 480 AlaTyrHisLys ArgGlyAla ProArgHisLys MetValMet AlaVal ccattttatgca cgtacctgg attctcgag 510 ProPheTyrAla ArgThrTrp IleLeuGlu <210> 44 <211> 170 <212> PRT

<213> Dermatophagoides farinae <400> 44 Asp Met G1u His Phe Thr Gln His Lys Gly Asn Ala Lys Ala Met Ile Ala Val Gly Gly Ser Thr Met Ser Asp Gln Phe Ser Lys Thr Ala Ala Val Glu His Tyr Arg Glu Thr Phe Val Val Ser Thr Val Asp Leu Met Thr Arg Tyr Gly Phe Asp Gly Val Met Ile Asp Trp Ser Gly Met Gln AL-2-C4-PCT.ST25.txt Ala Lys Asp Ser Asp Asn Phe Ile Lys Leu Leu Asp Lys Phe Asp Glu Lys Phe Ala His Thr Ser Phe Val Met Gly Val Thr Leu Pro Ala Thr Ile Ala Ser Tyr Asp Asn Tyr Asn Ile Pro Ala Ile Ser Asn Tyr Val Asp Phe Met Asn Val Leu Ser Leu Asp Tyr Thr Gly Ser Trp Ala His Thr Val G1y His Ala Ser Pro Phe Pro Glu Gln Leu Lys Thr Leu G1u Ala Tyr His Lys Arg Gly Ala Pro Arg His Lys Met Va1 Met Ala Val Pro Phe Tyr Ala Arg Thr Trp Ile Leu Glu <210> 45 <211> 510 <212> DNA
<213> Dermatophagoicles farinae <400>

ctcgagaatccaggtacgtgcataaaatggtacagccatgaccatcttatgacgtggagc60 gcctcgtttgtggtaagcttctagcgttttgagttgttcaggaaacggagaagcatgacc120 gaccgtatgggcccatgatccagtgtaatccagactaagcacgttcataaaatcgacata180 gttggagatggcaggaatgttatagttatcgtatgatgcgatcgttgccggcaaggtaac240 acccatcacaaacgaggtgtgagcaaacttttcgtcgaatttgtccaacaatttaatgaa300 attatcactatctttggcttgcatgccagaccaatcaatcatgacaccatcgaaaccata360 acgagtcataagatcaactgtgctaacaacaaacgtttcccgataatgttctaccgctgc420 agtcttggaaaattgatcggacatagtcgaaccaccgacggcgatcatggctttggcgtt480 gcccttatgttgtgtaaaatgttccatatc 510 <210> 46 <211> 25 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic Primer <220>
<221> misc feature AL-2-C4-PCT.ST25.txt <222> (15)..(15) <223> n = a, c, t, or g at position 15 <400> 46 gaaccaaaaa chgtntgyta ytayg 25 <210> 47 <211> 17 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic Primer <400> 47 gtaaaacgac ggccagt 17 <210> 48 <211> 29 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic Primer <400> 48 gatatggaac atttyachca acayaargg 29 <210> 49 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> Synthetic Primer <400> 49 gtaatacgac tcactatagg gc 22 <210> 50 <211> 1445 <212> DNA
<213> Dermatophagoides farinae <220>
<221> CDS
<222> (14)..(1399) <223>
<400> 50 atcccaaata aaa atg act cga ttc tct ttg act gta ttg gcc gta ctt 49 Met Thr Arg Phe Ser Leu Thr Val Leu Ala Val Leu gcc get tgt ttc ggt tca aat att cgt ccg aat gtg gca act ttg gaa 97 Ala Ala Cys Phe Gly Ser Asn Ile Arg Pro Asn Val Ala Thr Leu Glu AL-2-C4-PCT.ST25.txt cctaaa actgtatgt tactatgaa tcttgggta cattggcgc caaggt 145 ProLys ThrValCys TyrTyrGlu SerTrpVal HisTrpArg GlnGly gaaggc aaaatggat cccgaagac atagataca tcgttgtgt actcac 193 GluGly LysMetAsp ProGluAsp IleAspThr SerLeuCys ThrHis attgtc tactcttat ttcggcatt gatgetgcc actcatgag attaaa 241 IleVal TyrSerTyr PheGlyIle AspAlaAla ThrHisGlu IleLys ctattg gatgaatat cttatgaaa gatttacat gacatggaa catttc 289 LeuLeu AspGluTyr LeuMetLys AspLeuHis AspMetGlu HisPhe acgcag cataagggc aacgccaaa gccatgatc gccgtcggt ggttcg 337 ThrGln HisLysGly AsnAlaLys AlaMetIle AlaValGly GlySer actatg tccgatcaa ttttccaag actgcagcg gtagaacat tatcgg 385 ThrMet SerAspGln PheSerLys ThrAlaAla ValGluHis TyrArg gaaacg tttgttgtt agcaca gttgatcttatg actcgttat ggtttc 433 GluThr PheValVal SerThr ValAspLeuMet ThrArgTyr GlyPhe gatggt gtcatgatt gattgg tctggcatgcaa gccaaagat agtgat 481 AspGly ValMetIle AspTrp SerGlyMetGln AlaLysAsp SerAsp aatttc attaaattg ttggac aaattcgacgaa aagtttget cacacc 529 AsnPhe IleLysLeu LeuAsp LysPheAspGlu LysPheAla HisThr tcgttt gtgatgggt gttacc ttgccggcaacg atcgcatca tacgat 577 SerPhe ValMetGly ValThr LeuProAlaThr IleAlaSer TyrAsp aactat aacattcct gccatc tccaactatgtc gattttatg aacgtg 625 AsnTyr AsnIlePro AlaIle SerAsnTyrVal AspPheMet AsnVal cttagt ctggattac actgga tcatgggcccat acggtcggt catget 673 LeuSer LeuAspTyr ThrGly SerTrpAlaHis ThrValGly HisAla tctccg tttcctgaa caactc aaaacgctagaa gettaccac aaacga 721 SerPro PheProGlu GlnLeu LysThrLeuGlu AlaTyrHis LysArg ggcget ccacgtcat aagatg gtcatggetgta ccattttat gcacgt 769 GlyAla ProArgHis LysMet ValMetAlaVal ProPheTyr AlaArg acctgg attctcgag aaaatg aacaaacaggac attggcgat aaaget 817 ThrTrp IleLeuGlu LysMet AsnLysGlnAsp IleGlyAsp LysAla agtgga ccaggccca cgaggt cagtttacacag actgatggt ttcctt 865 SerGly ProGlyPro ArgGly GlnPheThrGln ThrAspGly PheLeu AL-2-C4-PCT.ST25.txt agctacaacgaa ttgtgcgttcag attcaggcc gaaacgaat gcattc 913 SerTyrAsnGlu LeuCysValGln IleGlnAla GluThrAsn AlaPhe accattactcgt gatcatgataat accgcaatt tacgetgtc tatgtg 961 ThrIleThrArg AspHisAspAsn ThrAlaIle TyrAlaVal TyrVal catagcaaccat gcagaatggatc tctttcgaa gaccgacat acactt 1009 HisSerAsnHis AlaGluTrpIle SerPheGlu AspArgHis ThrLeu ggtgaaaaagca aaaaacataacc caacaagga tatgetgga atgtca 1057 GlyGluLysAla LysAsnIleThr GlnGlnGly TyrAlaGly MetSer gtctacacattg tccaacgaagat gtgcacggc gtttgtggt gataaa 1105 ValTyrThrLeu SerAsnGluAsp ValHisGly ValCysGly AspLys aaccctttgttg catgetatccaa tcgaactat tatcatggc gtggta 1153 AsnProLeuLeu HisAlaIleGln SerAsnTyr TyrHisGly ValVal accgaaccgacc gtcgttacactt cctccagtc acacataca acagaa 1201 ThrGluProThr ValValThrLeu ProProVal ThrHisThr ThrGlu catgtgaccgat ataccaggcgtg tttcattgc catgaagaa ggattc 1249 HisValThrAsp IleProGlyVal ,PheHisCys HisGluGlu GlyPhe ttccgcgataag acctattgtgcc acatactac gaatgcaaa aaaggc 1297 PheArgAspLys ThrTyrCysAla ThrTyrTyr GluCysLys LysGly gattttggactg gagaaaaccgtg catcattgt gccaatcac ttacag 1345 AspPheGlyLeu GluLysThrVal HisHisCys AlaAsnHis LeuGln gcatttgacgaa gtaagtcggaca tgtattgat cataccaaa ataccc 1393 AlaPheAspGlu ,ValSerArgThr CysIleAsp HisThrLys IlePro ggttgttgaatacaaa taaaattaca aaaaaa 1445 atcactttaa aaaaaaaaaa GlyCys .

<210> 51 <211> 462 <212> PRT

<213> Dermatophago idesfarinae <400> 51 Met Thr Arg Phe Ser Leu Thr Val Leu Ala Val Leu Ala Ala Cys Phe Gly Ser Asn Ile Arg Pro Asn Val Ala Thr Leu Glu Pro Lys Thr Val AL-2-C4-PCT.ST25.txt Cys Tyr Tyr Glu Ser Trp Val His Trp Arg Gln Gly Glu Gly Lys Met Asp Pro Glu Asp Ile Asp Thr Ser Leu Cys Thr His Ile Val Tyr Ser Tyr Phe Gly Ile Asp Ala Ala Thr His Glu Ile Lys Leu Leu Asp Glu Tyr Leu Met Lys Asp Leu His Asp Met Glu His Phe Thr Gln His Lys Gly Asn Ala Lys Ala Met Tle Ala Val Gly Gly Ser Thr Met Ser Asp Gln Phe Ser Lys Thr Ala Ala Val Glu His Tyr Arg Glu Thr Phe Val 115 120 l25 Val Ser Thr Va1 Asp Leu Met Thr Arg Tyr Gly Phe Asp Gly Val Met Ile Asp Trp Ser Gly Met Gln Ala Lys Asp Ser Asp Asn Phe Ile Lys 145 l50 155 160 Leu Leu Asp Lys Phe Asp Glu Lys Phe Ala His Thr Ser Phe Val Met Gly Val Thr Leu Pro Ala Thr Ile Ala Ser Tyr Asp Asn Tyr Asn Tle Pro Ala Ile Ser Asn Tyr Val Asp Phe Met Asn Val Leu Ser Leu Asp Tyr Thr Gly Ser Trp Ala His Thr Val Gly His'Ala Ser Pro Phe Pro Glu G1n Leu Lys Thr Leu Glu Ala Tyr His Lys Arg Gly Ala Pro Arg His Lys Met Val Met Ala Val Pro Phe Tyr Ala Arg Thr Trp Ile Leu Glu Lys Met Asn Lys Gln Asp Ile Gly Asp Lys Ala Ser Gly Pro Gly Pro Arg Gly Gln Phe Thr Gln Thr Asp Gly Phe Leu Ser Tyr Asn Glu AL-2-C4-PCT.ST25.txt Leu Cys Val Gln Ile Gln Ala Glu Thr Asn Ala Phe Thr Ile Thr Arg Asp His Asp Asn Thr Ala Ile Tyr Ala Val Tyr Val His Ser Asn His 305 310 315 ' 320 Ala Glu Trp Ile Ser Phe Glu Asp Arg His Thr Leu Gly Glu Lys Ala Lys Asn Ile Thr Gln Gln Gly Tyr Ala Gly Met Ser Val Tyr Thr Leu Ser Asn Glu Asp Val His Gly Va1 Cys Gly Asp Lys Asn Pro Leu Leu His Ala Ile Gln Ser Asn Tyr Tyr His Gly Val Val Thr Glu Pro Thr Val Val Thr Leu Pro Pro Val Thr His Thr Thr Glu His Val Thr Asp Ile Pro Gly Val Phe His Cys His Glu Glu Gly Phe Phe Arg Asp Lys Thr Tyr Cys A1a Thr Tyr Tyr Glu Cys Lys Lys Gly Asp Phe Gly Leu Glu Lys Thr Val His His Cys Ala Asn His Leu Gln Ala Phe Asp Glu Val Ser Arg Thr Cys Ile Asp His Thr Lys Ile Pro Gly Cys <210> 52 <211> 1445 <212> DNA
<213> Dermatophagoides farinae <400> 52 tttttttttt ttttttttaa agtgattgta attttatttg tattcaacaa ccgggtattt 60 tggtatgatc aatacatgtc cgacttactt cgtcaaatgc ctgtaagtga ttggcacaat 120 gatgcacggt tttctccagt ccaaaatcgc cttttttgca ttcgtagtat gtggcacaat 180 aggtcttatc gcggaagaat ccttcttcat ggcaatgaaa cacgcctggt atatcggtca 240 catgttctgt tgtatgtgtg actggaggaa gtgtaacgac ggtcggttcg gttaccacgc 300 catgataata gttcgattgg atagcatgca acaaagggtt tttatcacca caaacgccgt 360 gcacatcttc gttggacaat gtgtagactg acattccagc atatccttgt tgggttatgt 420 AL-2-C4-PCT.ST25.txt tttttgctttttcaccaagtgtatgtcggtcttcgaaagagatccattctgcatggttgc480 tatgcacatagacagcgtaaattgcggtattatcatgatcacgagtaatggtgaatgcat540 tcgtttcggcctgaatctgaacgcacaattcgttgtagctaaggaaaccatcagtctgtg600 taaactgacctcgtgggcctggtccactagctttatcgccaatgtcctgtttgttcattt660 tctcgagaatccaggtacgtgcataaaatggtacagccatgaccatcttatgacgtggag720 cgcctcgtttgtggtaagcttctagcgttttgagttgttcaggaaacggagaagcatgac780 cgaccgtatgggcccatgatccagtgtaatccagactaagcacgttcataaaatcgacat840 agttggagatggcaggaatgttatagttatcgtatgatgcgatcgttgccggcaaggtaa900 cacccatcacaaacgaggtgtgagcaaacttttcgtcgaatttgtccaacaatttaatga960 aattatcactatctttggcttgcatgccagaccaatcaatcatgacaccatcgaaaccat1020 aacgagtcataagatcaactgtgctaacaacaaacgtttcccgataatgttctaccgctg1080 cagtcttggaaaattgatcggacatagtcgaaccaccgacggcgatcatggctttggcgt1140 tgcccttatgctgcgtgaaatgttccatgtcatgtaaatctttcataagatattcatcca1200 atagtttaatctcatgagtggcagcatcaatgccgaaataagagtagacaatgtgagtac1260 acaacgatgtatctatgtcttcgggatccattttgccttcaccttggcgccaatgtaccc1320 aagattcatagtaacatacagttttaggttccaaagttgccacattcggacgaatatttg1380 aaccgaaacaagcggcaagtacggccaatacagtcaaagagaatcgagtcatttttattt1440 gggat <210> 53 <211> 1386 <212> DNA
<213> Dermatophagoides farinae <400>

atgactcgattctctttgactgtattggccgtacttgccgcttgtttcggttcaaatatt60 cgtCCgaatgtggcaactttggaacctaaaactgtatgttactatgaatcttgggtacat120 tggcgccaaggtgaaggcaaaatggatcccgaagacatagatacatcgttgtgtactcac180 attgtctactcttatttcggcattgatgctgccactcatgagattaaactattggatgaa240 tatcttatgaaagatttacatgacatggaacatttcacgcagcataagggcaacgccaaa300 gccatgatcgccgtcggtggttcgactatgtccgatcaattttccaagactgcagcggta360 gaacattatcgggaaacgtttgttgttagcacagttgatcttatgactcgttatggtttc420 gatggtgtcatgattgattggtctggcatgcaagccaaagatagtgataatttcattaaa480 ttgttggacaaattcgacgaaaagtttgctcacacctcgtttgtgatgggtgttaccttg540 ccggcaacgatcgcatcatacgataactataacattcctgccatctccaactatgtcgat600 tttatgaacgtgcttagtctggattacactggatcatgggcccatacggtcggtcatgct660 AL-2-C4-PCT.ST25.txt tctccgtttcctgaacaactcaaaacgctagaagcttaccacaaacgaggcgctccacgt720 cataagatggtcatggctgtaccattttatgcacgtacctggattctcgagaaaatgaac780 aaacaggacattggcgataaagctagtggaccaggcccacgaggtcagtttacacagact840 gatggtttccttagctacaacgaattgtgcgttcagattcaggccgaaacgaatgcattc900 accattactcgtgatcatgataataccgcaatttacgctgtctatgtgcatagcaaccat960 gcagaatggatctctttcgaagaccgacatacacttggtgaaaaagcaaaaaacataacc1020 caacaaggatatgctggaatgtcagtctacacattgtccaacgaagatgtgcacggcgtt1080' tgtggtgataaaaaccctttgttgcatgctatccaatcgaactattatcatggcgtggta1140 accgaaccgaccgtcgttacacttcctccagtcacacatacaacagaacatgtgaccgat1200 ataccaggcgtgtttcattgccatgaagaaggattcttccgcgataagacctattgtgcc1260 acatactacgaatgcaaaaaaggcgattttggactggagaaaaccgtgcatcattgtgcc1320 aatcacttacaggcatttgacgaagtaagtcggacatgtattgatcataccaaaataccc1380 ggttgt 1386 <210>

<211>

<212>
DNA

<213>
Dermatophagoides farinae <400>

acaaccgggtattttggtatgatcaatacatgtccgacttacttcgtcaaatgcctgtaa60 gtgattggcacaatgatgcacggttttctccagtccaaaatcgccttttttgcattcgta120 gtatgtggcacaataggtcttatcgcggaagaatccttcttcatggcaatgaaacacgcc180 tggtatatcggtcacatgttctgttgtatgtgtgactggaggaagtgtaacgacggtcgg240 ttcggttaccacgccatgataatagttcgattggatagcatgcaacaaagggtttttatc300 accacaaacgccgtgcacatcttcgttggacaatgtgtagactgacattccagcatatcc360 ttgttgggttatgttttttgctttttcaccaagtgtatgtcggtcttcgaaagagatcca420 ttctgcatggttgctatgcacatagacagcgtaaattgcggtattatcatgatcacgagt480 aatggtgaatgcattcgtttcggcctgaatctgaacgcacaattcgttgtagctaaggaa540 accatcagtctgtgtaaactgacctcgtgggcctggtccactagctttatcgccaatgtc600 ctgtttgttcattttctcgagaatccaggtacgtgcataaaatggtacagccatgaccat660 cttatgacgtggagcgcctcgtttgtggtaagcttctagcgttttgagttgttcaggaaa720 cggagaagcatgaccgaccgtatgggcccatgatccagtgtaatccagactaagcacgtt780 cataaaatcgacatagttggagatggcaggaatgttatagttatcgtatgatgcgatcgt840 tgccggcaaggtaacacccatcacaaacgaggtgtgagcaaacttttcgtcgaatttgtc900 AL-2-C4-PCT.ST25.txt caacaatttaatgaaattatcactatctttggcttgcatgccagaccaatcaatcatgac960 accatcgaaaccataacgagtcataagatcaactgtgctaacaacaaacgtttcccgata1020 atgttctaccgctgcagtcttggaaaattgatcggacatagtcgaaccaccgacggcgat1080 catggctttggcgttgcccttatgctgcgtgaaatgttccatgtcatgtaaatctttcat1140 aagatattcatccaatagtttaatctcatgagtggcagcatcaatgccgaaataagagta1200 gacaatgtgagtacacaacgatgtatctatgtcttcgggatccattttgccttcaccttg1260 gcgccaatgtacccaagattcatagtaacatacagttttaggttccaaagttgccacatt1320 cggacgaatatttgaaccgaaacaagcggcaagtacggccaatacagtcaaagagaatcg1380 agtcat 1386 <210> 55 <211> 1236 <212> DNA

<213> Dermatophagoides farinae <220>
<221> CDS
<222> (1)..(1236) <223>
<400>

actttggaacct aaaactgta tgttactat gaatcttgg gtacattgg 48 ThrLeuGluPro LysThrVal CysTyrTyr GluSerTrp ValHisTrp cgccaaggtgaa ggcaaaatg gatcccgaa gacatagat acatcgttg 96 ArgGlnGlyGlu GlyLysMet AspProGlu AspTleAsp ThrSerLeu tgtactcacatt gtctactct tatttcggc attgatget gccactcat 144 CysThrHisIle ValTyrSer TyrPheGly IleAspAla AlaThrHis gagattaaacta ttggatgaa tatcttatg aaagattta catgacatg 192 GluIleLysLeu LeuAspGlu TyrLeuMet LysAspLeu HisAspMet gaacatttcacg cagcataag ggcaacgcc aaagccatg atcgccgtc 240 GluHisPheThr GlnHisLys GlyAsnAla LysAlaMet IleAlaVal ggtggttcgact atgtccgat caattttcc aagactgca gcggtagaa 288 GlyGlySerThr MetSerAsp GlnPheSer LysThrAla AlaValGlu cattatcgggaa acgtttgtt gttagcaca gttgatctt atgactcgt 336 HisTyrArgGlu ThrPheVal ValSerThr ValAspLeu MetThrArg tatggtttcgat ggtgtcatg attgattgg tctggcatg caagccaaa 384 TyrGlyPheAsp GlyValMet IleAspTrp SerGlyMet GlnAlaLys gatagtgataat ttcattaaa ttgttggac aaattcgac gaaaagttt 432 AL-2-C4-PCT.ST25.txt AspSerAspAsn PheIleLys LeuLeuAsp LysPheAspGlu LysPhe getcacacctcg tttgtgatg ggtgttacc ttgccggcaacg atcgca 480 AlaHisThrSer PheValMet GlyValThr LeuProAlaThr IleAla tcatacgataac tataacatt cctgccatc tccaactatgtc gatttt 528 SerTyrAspAsn TyrAsnIle ProAlaIle SerAsnTyrVal AspPhe atgaacgtgctt agtctggat tacactgga tcatgggcccat acggtc 576 MetAsnValLeu SerLeuAsp TyrThrGly SerTrpAlaHis ThrVal ggtcatgettct ccgtttcct gaacaactc aaaacgctagaa gettac 624 GlyHisAlaSer ProPhePro GluGlnLeu LysThrLeuGlu AlaTyr cacaaacgaggc getccacgt cataagatg gtcatggetgta ccattt 672 HisLysArgGly AlaProArg HisLysMet ValMetAlaVal ProPhe tatgcacgtacc tggattctc gagaaaatg aacaaacaggac attggc 720 TyrAlaArgThr TrpIleLeu GluLysMet AsnLysGlnAsp IleGly gataaagetagt ggaccaggc ccacgaggt cagtttacacag actgat 768 AspLysAlaSer GlyProGly ProArgGly GlnPheThrGln ThrAsp ggtttccttagc tacaacgaa ttgtgcgtt cagattcaggcc gaaacg 816 GlyPheLeuSer TyrAsnGlu LeuCysVal GlnIleGlnAla GluThr aatgcattcacc attactcgt gatcatgat aataccgcaatt tacget 864 AsnAlaPheThr IleThrArg AspHisAsp AsnThrAlaIle TyrAla gtctatgtgcat agcaaccat gcagaatgg atctctttcgaa gaccga 912 ValTyrValHis SerAsnHis AlaGluTrp IleSerPheGlu AspArg catacacttggt gaaaaagca aaaaacata acccaaCaagga tatget 960 HisThrLeuGly GluLysAla LysAsnIle ThrGlnGlnGly TyrAla ggaatgtcagtc tacacattg tccaacgaa gatgtgcacggc gtttgt 1008 GlyMetSerVal TyrThrLeu SerAsnGlu AspValHisGly ValCys ggtgataaaaac cctttgttg catgetatc caatcgaactat tatcat 1056 GlyAspLysAsn ProLeuLeu HisAlaIle GlnSerAsnTyr TyrHis ggcgtggtaacc gaaccgacc gtcgttaca cttcctccagtc acacat 1104 GlyValValThr GluProThr ValVa1Thr LeuProProVal ThrHis acaacagaacat gtgaccgat ataccaggc gtgtttcattgc catgaa 1152 ThrThrGluHis ValThrAsp IleProGly ValPheHisCys HisGlu gaaggattcttc cgcgataag acctattgt gccacatactac gaatgc 1200 AL-2-C4-PCT.ST25.txt Glu Gly Phe Phe Ar,g Asp Lys Thr Tyr Cys Ala Thr Tyr Tyr Glu Cys aaa aaa ggC gat ttt gga ctg gag aaa acc gtg cat 1236 Lys Lys Gly Asp Phe Gly Leu Glu Lys Thr Val His i <210> 56 <211> 412 <212> PRT
<213> Dermatophagoides farinae <400> 56 Thr Leu Glu Pro Lys Thr Val Cys Tyr Tyr G1u Ser Trp Val His Trp Arg Gln Gly Glu Gly Lys Met Asp Pro Glu Asp Ile Asp Thr Ser Leu Cys Thr His Ile Val Tyr Ser Tyr Phe Gly Ile Asp Ala Ala Thr His Glu Tle Lys Leu Leu Asp Glu Tyr Leu Met Lys Asp Leu His Asp Met Glu His Phe Thr Gln His Lys Gly Asn Ala Lys Ala Met Ile Ala Va1 Gly Gly Ser Thr Met Ser Asp G1n Phe Ser Lys Thr Ala Ala Val Glu His Tyr Arg Glu Thr Phe Val Val Ser Thr Val Asp Leu Met Thr Arg Tyr Gly Phe Asp Gly Val Met Ile Asp Trp Ser Gly Met Gln Ala Lys Asp Ser Asp Asn Phe Ile Lys Leu Leu Asp Lys Phe Asp Glu Lys Phe Ala His Thr Ser Phe Val Met Gly Val Thr Leu Pro Ala Thr I1e A1a Ser Tyr Asp Asn Tyr Asn Ile Pro Ala I1e Ser Asn Tyr Val Asp Phe Met Asn Val Leu Ser Leu Asp Tyr Thr Gly Ser Trp Ala His Thr Val Gly His Ala Ser Pro Phe Pro Glu Gln Leu Lys Thr Leu Glu Ala Tyr AL-2-C4-PCT.ST25.txt His Lys Arg Gly Ala Pro Arg His Lys Met Val Met Ala Val Pro Phe Tyr Ala Arg Thr Trp Ile Leu Glu Lys Met Asn Lys Gln Asp Ile Gly Asp Lys Ala Ser G1y Pro Gly Pro Arg Gly Gln Phe Thr Gln Thr Asp Gly Phe Leu Ser Tyr Asn Glu Leu Cys Val Gln Ile Gln Ala Glu Thr Asn Ala Phe Thr Ile Thr Arg Asp His Asp Asn Thr Ala I1e Tyr Ala Val Tyr Val His Ser Asn His Ala Glu Trp Ile Ser Phe Glu Asp Arg His Thr Leu Gly Glu Lys Ala Lys Asn Ile Thr Gln Gln Gly Tyr Ala Gly Met Ser Val Tyr Thr Leu Ser Asn Glu Asp Val His Gly Val Cys Gly Asp Lys Asn Pro Leu Leu His Ala Ile Gln Ser Asn Tyr Tyr His G1y Val Val Thr Glu Pro.Thr Val Val Thr Leu Pro Pro Val Thr His Thr Thr G1u His Val Thr Asp Ile Pro Gly Val Phe His Cys His Glu Glu Gly Phe Phe Arg Asp Lys Thr Tyr Cys Ala Thr Tyr Tyr Glu Cys Lys Lys Gly Asp Phe Gly Leu Glu Lys Thr Va1 His <210> 57 <211> 1236 <212> DNA
<213> Dermatophagoides farinae <400> 57 atgcacggtt ttctccagtc caaaatcgcc ttttttgcat tcgtagtatg tggcacaata 60 ggtcttatcg cggaagaatc cttcttcatg gcaatgaaac acgcctggta tatcggtcac 120 AL-2-C4-PCT.ST25.txt atgttctgttgtatgtgtgactggaggaagtgtaacgacggtcggttcggttaccacgcc180 atgataatagttcgattggatagcatgcaacaaagggtttttatcaccacaaacgccgtg240 cacatcttcgttggacaatgtgtagactgacattccagcatatccttgttgggttatgtt300 ttttgctttttcaccaagtgtatgtcggtcttcgaaagagatccattctgcatggttgct360 atgcacatagacagcgtaaattgcggtattatcatgatcacgagtaatggtgaatgcatt420 cgtttcggcctgaatctgaacgcacaattcgttgtagctaaggaaaccatcagtctgtgt480 aaactgacctcgtgggcctggtccactagctttatcgccaatgtcctgtttgttcatttt540 ctcgagaatccaggtacgtgcataaaatggtacagccatgaccatcttatgacgtggagc600 gcctcgtttgtggtaagcttctagcgttttgagttgttcaggaaacggagaagcatgacc660 gaccgtatgggcccatgatccagtgtaatccagactaagcacgttcataaaatcgacata720 gttggagatggcaggaatgttatagttatcgtatgatgcgatcgttgccggcaaggtaac780 acccatcacaaacgaggtgtgagcaaacttttcgtcgaatttgtccaacaatttaatgaa840 attatcactatctttggcttgcatgccagaccaatcaatcatgacaccatcgaaaccata900 acgagtcataagatcaactgtgctaacaacaaacgtttcccgataatgttCtaCCgCtgC960 agtcttggaaaattgatcggacatagtcgaaccaccgacggcgatcatggctttggcgtt1020 gcccttatgctgcgtgaaatgttccatgtcatgtaaatctttcataagatattcatccaa1080 tagtttaatctcatgagtggcagcatcaatgccgaaataagagtagacaatgtgagtaca1140 caacgatgtatctatgtcttcgggatccattttgccttcaccttggcgccaatgtaccca1200 agattcatagtaacatacagttttaggttccaaagt 1236

Claims

What is claimed is:

1. An isolated nucleic acid molecule selected from the group consisting of:

(a) 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 1X SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid molecule comprising 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 of SEQ ID NO:33 and a complement thereof; and (b) a nucleic acid molecule comprising a fragment of any of said nucleic acid molecules of (a) wherein said fragment comprises at least about nucleotides.

2. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence that encodes a Der HMW-map protein.

3. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule is selected from the group consisting of nDerf98 1752, nDerf98 1665, nDerf98 1608, nDerp98 1621, nDerp98 1527, nDerp98 1470, and nDerf60 510.

4. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule is selected from the group consisting of:

(a) a nucleic acid molecule comprising 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 (b) a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule of (a).

5. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule is selected from the group consisting of:

(a) a nucleic acid molecule comprising a nucleic acid sequence that encodes a protein having an amino acid sequence selected from the group consisting 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, and SEQ ID NO:44; and (b) a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule encoding a protein having an amino acid sequence of (a).

6. A recombinant molecule comprising a nucleic acid molecule according to Claims 1-5.

7. A recombinant virus comprising a nucleic acid molecule according to Claims 1-5.

8. A recombinant cell comprising a nucleic acid molecule according to Claims 1-5.

9. An isolated protein encoded by a nucleic acid molecule selected from the group consisting of:

(a) 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 1X SSC and 0% formamide, at a temperature of about 50°C, to a nucleic acid molecule comprising 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 (b) a nucleic acid molecule comprising a fragment of any of said nucleic acid molecules of (a), wherein said fragment comprises at least about nucleotides.

10. The protein of Claim 9, wherein said protein, when administered to an animal, elicits an immune response against a Der HMW-map protein.

11. The protein of Claim 9, wherein said protein is selected from the group consisting of:

(a) a protein encoded by a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of: 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 the coding strand of a nucleic acid sequence encoding a protein comprising the amino acid sequence SEQ ID NO:33; and (b) a protein encoded by a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule comprising any of said nucleic acid molecules of (a).

12. The protein of Claim 9, wherein said protein is selected from the group consisting of:
(a) a protein comprising an amino acid sequence selected from the group consisting 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 117 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 SEQ ID NO:44; and (b) a protein encoded by an allelic variant of a nucleic acid molecule encoding a protein comprising any of said amino acid sequences of (a).

13. An isolated antibody that selectively binds to a protein as set forth in Claim 9.

14. The protein of Claim 9, wherein said protein selectively binds to IgE.

15. The protein of Claim 9, wherein said protein comprises an epitope having at least one identifying characteristic selected from the group consisting of:

(a) said epitope is resistant to .beta.-elimination of peptides;

(b) said epitope is resistant to Proteinase-K digestion; and (c) said epitope is reactive to a test designed to detect glycosylated proteins, wherein said epitope binds to an IgE selected from the group consisting of canine IgE from dogs allergic to mites and feline IgE from cats allergic to mites.

16. A therapeutic composition for treating an allergic response to a mite, said therapeutic composition comprising a desensitizing compound selected from the group consisting of:

(a) an isolated protein according to Claims 9-12;
(b) a mimetope of said mite allergenic protein;
(c) a mutein of said mite allergenic protein;
(d) an isolated nucleic acid molecule according to Claims 1-5;
(e) an antibody to said mite allergic protein; and (f) an inhibitor of binding of said mite allergic protein to IgE.

17. The composition of Claim 16, wherein said composition further comprises a component selected from the group consisting of an excipient, an adjuvant, and a carrier.

18. The composition of Claim 16, wherein said desensitizing compound is a naked nucleic acid molecule.

19. An assay kit for testing if an animal is susceptible to or has an allergic response to a mite, said kit comprising:

(a) an isolated protein according to Claims 9-12; and (b) a means for determining if said animal is susceptible to or has said allergic response, wherein said means comprises use of said protein to identify animals susceptible to or having allergic responses to mites.

20. A method to identify an animal susceptible to or having an allergic response to a mite, said method comprising:

(a) contacting an isolated protein according to Claims 9-12 with antibodies of an animal; and (b) determining immunocomplex formation between said protein and said antibodies, wherein formation of said immunocomplex indicates that said animal is susceptible to or has said allergic response.

21. A method to desensitize a host animal to an allergic response to a mite, said method comprising administering to said animal a therapeutic composition according to Claim 16.

22. The method of Claim 21, wherein said protein comprises an amino acid sequence selected from the group consisting 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 117 NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID
NO:35, SEQ ID NO:38, SEQ ID NO:41, and SEQ ID NO:44.

23. The method of Claim 21, wherein said therapeutic composition further comprises a component selected from the group consisting of an excipient, an adjuvant and a carrier.

24. A method to produce a mite allergenic protein, said method comprising culturing a cell transformed with 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 1X 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, wherein said fragment comprises at least about 15 nucleotides.

25. A reagent comprising a non-proteinaceous epitope having at least one identifying characteristic selected from the group consisting of:
(a) said epitope is resistant to .beta.-elimination of peptides;
(b) said epitope is resistant to Proteinase-K digestion; and (c) said epitope is reactive to a test designed to detect glycosylated proteins, wherein said epitope binds to an IgE selected from the group consisting of canine IgE
from dogs allergic to mites and feline IgE from cats allergic to mites.

26. An isolated antibody that selectively binds to an epitope as set forth in Claim 25.

27. A therapeutic composition for treating an allergic response to a mite, said therapeutic composition comprising a desensitizing compound comprising the reagent of Claim 25.

28. An assay kit for testing if an animal is susceptible to or has an allergic response to a mite, said kit comprising the reagent of Claim 25 and a means for determining if said animal is susceptible to or has said allergic response, wherein said means comprises use of said reagent to identify animals susceptible to or having allergic responses to mites.

29. A method to identify an animal susceptible to or having an allergic response to a mite, said method comprising:

(a) contacting the reagent of Claim 25 with antibodies of an animal;
and (b) determining immunocomplex formation between said reagent and said antibodies, wherein formation of said immunocomplex indicates that said animal is susceptible to or has said allergic response.

30. A method to desensitize a host animal to an allergic response to a mite, said method comprising administering to said animal a therapeutic composition comprising a desensitizing compound comprising the reagent of Claim 25.