AU783254B2 - Immunogenic proteins from the domestic mite, blomia tropicalis - Google Patents

Description

Received 12 December 2001 r :,op:a lrWpcuc uN233"0bI WW e 2/,4,e.l12MI1 -1- NOVEL THERAPEUTIC MOLECULES AND USES THEREFOR FIELD OF THE INVENTION The present invention relates generally to novel protein molecules and to derivatives, homologs, analogues, chemical equivalents and mimetics thereof capable of inducing, upregulating or otherwise facilitating the induction of an immune response to a mite and, more particularly, a mite from the family Glycyphagidae. These mites include dust mites and storage mites. The present invention also contemplates genetic sequences encoding said protein molecules and derivatives, homologs, analogues, chemical equivalents and mimetics thereof. The present invention further provides genetic vaccines and other compositions comprising nucleic acid molecules. The molecules of the present invention are useful, inter alia, in a range of therapeutic, prophylactic and diagnostic applications.

BACKGROUND OF THE INVENTION Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia or any other country.

House dust or storage mite is one of the most important sources of indoor allergens and their role in triggering allergic diseases has been established Besides two predominant mite species, Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae Blomia tropicalis (Bt) is equally important in sensitizing allergic patients in tropical and subtropical areas of the world. Although allergens of B. tropicalis are considered as a major component of dust in Singapore there is no significant differences in sensitization pattern of mite sensitized population here However, the importance of including B. tropicalis allergens in the diagnostic and therapeutic reagents is Received 12 December 2001 P:patspum2337OLa=muLink a l -am mn1-o.12/II1 -2readily shown by a cross-reactivity study which showed there is very limited crossreactivity between these two family of mites This pyroglyphid mite species has been well documented to induce allergic asthma and allergic rhinitis Epidemiological studies clearly indicated that Dp and Bt mites from the Pyroglyphidae and Glycyphagidae respectively, are the most common and important clinical mite species in the tropical and sub-tropical regions of the world (11,12,13). Thus, there are allergens which remain unknown and have to be characterized in B. tropicalis.

Studies on the allergenecity of B. tropicalis revealed the presence of more than allergens. Crossed radioimmunoelectrophoresis (CRIE) studies identified 21 different species-specific IgE binding-antigens in B. tropicalis (14) while immunoblotting techniques were able to reveal the presence of 25 allergens with molecular weights ranging from 11 to 85 kD Despite these initial findings, however, only three published Bt molecules purported to be allergens had been previously identified and characterized. Blo t 5, the first Bt allergen to be cloned with 40% amino acid homology to Der p 5, contains 432 bp open reading frame To date, Blo t 5 is the only major allergen isolated from B.

tropicalis with 70% IgE reactivity to mite allergic sera (16,17). Two other Bt allergens, Bt 6 (now identified as Btl3) and Bt Ila (now identified as Btl2), exhibited human IgE reactivity of 11% (18) and 50% respectively. Bt 13 is composed of 934 bp with 390 bp open reading frame which codes for a 130 amino acid allergen with a molecular weight of 14.8 kD On the other hand, Bt 12 is composed of 582 bp with 432 bp reading frame coding for a 20-residue putative signal peptide and a 124-residue mature protein with an estimated molecular weight of 14.206 kD Thus far, the reported Bt allergens are low molecular weight proteins. Recent studies in other species of mites demonstrated the presence of high molecular weight allergens with significant IgE-binding properties from mite sensitive patients Df 11 isolated from Dermatophagoides farinae is a 98 kD paramyosin allergen with a human IgE reactivity of 82% (21) while Mag 3 (M-177) isolated from Dermatophagoides pteronyssinus is a 177 kD allergen with a human IgE reactivity of 70% (22).

Received 12 December 2001 -3- To date, crude mite extract has been the sole diagnostic and therapeutic reagent for mite allergies. Due to poor extraction methodology and the incidence of protein degredation, it has not been possible to determine a complete profile of the immunogenic regions and/or specific epitopes of the dust or storage mite. This has resulted in poor diagnosis and immunotherapy of conditions such as mite induced allergies. Accordingly, there is a need to identify immunogenic regions and/or epitopes of the dust or storage mite in order to facilitate better diagnosis and immunotherapy of conditions related to the dust or storage mite, such as allergies.

In work leading up to the present invention, the inventors have studied the domestic mite tropicalis) and have identified and sequenced novel immunogenic protein molecules.

The identification of these molecules facilitates, inter alia, the development of more efficacious clinical reagents for the treatment and diagnosis of conditions involving the induction of an immune response to the mite, such as allergies. The allergens identified in accordance with the present invention are referred to herein as "Bt" allergens. Specific Bt allergens are "Btl "BtlO", "Bt5" and "BtA2". Insofar as "Bt5" is part of the invention, it is a polymorphic variant of the Bt5, as published by Arruda et al. and consisting of the amino acid sequence and corresponding nucleotide sequence set forth in SEQ ID NO:36.

Received 12 December 2001 poiJm~pCaf3l3t=L~bbc* eaCa+llfc9ftS~a0l"jlx/\I\ -4- SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1, <400>2, etc. A sequence listing is provided after the claims.

One aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from B. tropicalis or a derivative, homologue or mimetic of said protein allergen.

Another aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is selected from Btl 1, BtlO and BtA2, each as herein defined, or polymorphic variants thereof and a polymorphic variant of as herein defined.

A further aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encodingor complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is Btl 1 having an amino acid sequence as set forth in any one of SEQ ID NO:2 to SEQ ID NO: 14 or a derivative, homologue or mimetic thereof or having at least about 45% or greater similarity to an amino acid sequence set forth in in SEQ ID NO:2 to SEQ ID NO:14.

A L JI WW VVI V a& I Received 12 December 2001 r.opa-.1 ptna337 m\ l.H o m imil2lc pliM B l 12inda- /I2MI Still another aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is Btll wherein said nucleotide sequence is as substantially set forth in SEQ ID NO:1 or SEQ ID NO: 2 or SEQ ID NO:13 or a derivative, homologue or mimetic thereof or capable of hybridizing to SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO: 13 or a complementary form thereof under low stringency conditions.

Yet another aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is BtlO having an amino acid sequence as substantially set forth in SEQ ID NO:29 or SEQ ID NO: 31 or a derivative, homologue or mimetic thereof having at least about 45% similarity to amino acid sequences set forth in SEQ ID NO:29 or SEQ ID NO:31.

Still another aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is BtlO wherein said nucleotide sequence is as substantially set forth in SEQ ID NO:28 or SEQ ID NO: 30 or a nucleotide sequence having at least 45% similarity to SEQ ID NO:28 or SEQ ID NO:30 or a nucleotide sequence capable of hybridizing to SEQ ID NO:28 or SEQ ID NO:30 or a complementary form thereof under low stringency conditions.

Even still another aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is a polymorphic variant of Bt5 having an amino acid sequence as set forth in any one of SEQ Received 12 December 2001 PopaW tOL&daci. n jifmdxIa -6- ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71 or a derivative, homologue or mimetic thereof or having at least about 45% similarity to an amino acid sequence set forth in any one of SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71.

Even yet another aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is a polymorphic variant of Bt5 wherein said nucleotide sequence is substantially as set forth in any one of SEQ ID NO:38 to SEQ ID NO:40 or SEQ ID NO:55 to SEQ ID NO:67 or a derivative, homologue or mimetic thereof or a nucleotide sequence having at least about 45% similarity to SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71 or a nucleotice sequence capable of hybridizing to any one of SEQ ID NO:38 to SEQ ID NO:40 or SEQ ID NO:55 to SEQ ID NO:67 or a complementary form thereof under low stringency conditions.

Another aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is BtA2 having an amino acid sequence as set forth in SEQ ID NO:75 or a derivative, homologue or mimetic thereof or having at least about 45% or greater similarity to an amino acid sequence set forth in SEQ ID A further aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is BtA2 wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:74 or a derivative, homologue or mimetic thereof or a nucleotide sequence having at least about 45% similarity to SEQ ID NO:74 or a nucleotide sequence capable of hybridizing to SEQ ID NO:74.

Received 12 December 2001 -7- Yet another aspect of the present invention contemplates an isolated protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is selected from Btl BtlO or BtA2, each as herein defined, or polymorphic variants thereof and a polymorphic variant of Bt5 as herein defined.

Still another aspect of the present invention contemplates an isolated protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is Btl 1, said amino acid sequence substantially as set forth in SEQ ID NO:3 to SEQ ID NO:12 or SEQ ID NO:14 or a derivative, homologue or mimetic thereof or having at least about 45% similarity to an amino acid sequence set forth in SEQ ID NO:3 to SEQ ID NO:12 or SEQ ID NO:14.

Even still another aspect of the present invention contemplates an isolated protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is Btl 1, said amino acid sequence encoded by the nucleotide sequene set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13 or a derivative, homologue or mimetic thereof or a nucleotide sequence capable of hybridizing to SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:13 or a complementary form thereof under low stringency conditions.

Even still another aspect of the present invention contemplates an isolated protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is BtlO, said amino acid sequence substantially as set forth in SEQ ID NO:28 or SEQ ID NO:31 or a derivative, homologue or mimetic thereof or having at least about 45% similarity to an amino acid sequence set forth in SEQ ID NO:28 or SEQ ID NO:31.

Even yet another another aspect of the present invention contemplates an isolated protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is BtlO, said amino acid sequence encoded by the nucleotide Received 12 December 2001 Pa.VarA PdZ337MbL r I2/1 I -8sequene set forth in SEQ ID NO:28 or SEQ ID NO:30 or a derivative, homologue or mimetic thereof or a nucleotide sequence capable of hybridizing to SEQ ID NO:28 or SEQ ID NO:30 or a complementary form thereof under low stringency conditions.

Another another aspect of the present invention contemplates an isolated protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is a polymorphic variant of Bt5, said amino acid sequence substantially as set forth in SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71 or a derivative, homologue or mimetic thereof or having at least about 45% similarity to an amino acid sequence set forth in SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71.

A further aspect another aspect of the present invention contemplates an isolated protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is a polymorphic variant of Bt5, said amino acid sequence encoded by the nucleotide sequene set forth in SEQ ID NO:38 to SEQ ID NO:40 or SEQ ID NO:55 to SEQ ID NO:67 or a derivative, homologue or mimetic thereof or a nucleotide sequence capable of hybridizing to SEQ ID NO:38 to SEQ ID NO:40 or SEQ ID NO:55 to SEQ ID NO:67 or a complementary form thereof under low stringency conditions.

Yet another another aspect of the present invention contemplates an isolated protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is BtA2, said amino acid sequence substantially as set forth in SEQ ID NO:75 or a derivative, homologue or mimetic thereof or having at least about 45% similarity to an amino acid sequence set forth in SEQ ID Still another another aspect of the present invention contemplates an isolated protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is BtA2, said amino acid sequence encoded by the nucleotide sequene set forth in SEQ ID NO:74 or a derivative, homologue or mimetic thereof or a nucleotide sequence capable of hybridizing to SEQ ID NO:74 or a complementary form Received 12 December 2001 1rv.lT prfwi. vml7g f m I I rrrnnH^ up i .Ilila -9thereof under low stringency conditions.

Table 1 is a list of single and three letter abbreviations used throughout the specification.

Table 2 is a summary of amino acid and nucleotide sequence identifiers.

Table 3 summarizes the characteristics of the polymorphic residues found in Bt5 genes.

Table 4 is a summary of the results of human IgE plaque immunoassay (Example 13).

Table 5 is a summary of the results of dot blot immunoassay (Example 13).

Received 12 December 2001 TABLE I Single and three letter amino acid abbreviations Amino Acid Three-letter One-letter Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamnine Gin Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine The T Tryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X Received 12 December 2001 P.~op:%E)pwpat337=bLOU ioed. spwaw4'Wr o&r -2IIMI 11- TABLE 2 SEQUENCE IDENTIFIER DESCRIPTION SEQ ID NO: 1 Nucleotide sequence of clone encoding Btl 1 SEQ ID NO:2 Nucleotide and corresponding amino acid sequences of clone encoding Btl 1 SEQ ID NO:3 to SEQ ID NO: 12 Amino acid sequences encoded by SEQ ID NO:2 SEQ ID NO:13 Nucleotide sequence and corresponding amino acid sequence of Btl gene SEQ ID NO:14 Amino acid sequence of Btl 1 SEQ ID NO:15 Amino acid sequence of Dermatophagoides farinae paramyosin SEQ ID NO:16 Amino acid sequence of Drosophila melanogaster paramyosin SEQ ID NO:17 Amino acid sequence of Onchocerca volvulus paramyosin SEQ ID NO:18 Amino acid sequence of Caenorhabditis elegans paramyosin SEQ ID NO:19 T3 primer SEQ ID NO:20 T7 primer SEQ ID NO:21 Gene-specific primer 1 (GSP1) SEQ ID NO:22 Gene-specific primer 2 (GSP2) SEQ ID NO:23 Nested universal primer (NUP) SEQ ID NO:24 T7 primer SEQ ID NO:25 Btll CS2f primer SEQ ID NO:26 Btl1 R2 primer SEQ ID NO:27 Btl 1 polymorphic variant SEQ ID NO:28 Nucleotide sequence of clone encoding Btl0 SEQ ID NO:29 Amino acid sequence encoded by SEQ ID NO:28 SEQ ID NO:30 Nucleotide sequence of coding region of SEQ ID NO:28 SEQ ID NO:31 Amino acid sequence encoded by SEQ ID SEQ ID NO:32 Amino acid sequence of Group 10 allergen from Dermatophagoidesfarinae (Dfl 0) SEQ ID NO:33 Amino acid sequence of Group 10 allergen from Lepidoglyphus desructor (Lp Received 12 December 2001 Pop:c*uwWAp=3370LbkL 12/12101 12 SEQUENCE IDENTIFIER DESCRIPTION CTt rmK A Amino acid sequence of GrouplO allergen from Dermatophagoides pteronyssinus (Dp SEQ ID NO:35 Amino acid sequence of Group 10 allergen from Dermatophagoidespteronyssinus (Dp SEQ ID NO:36 Bt5 nucleotide sequence (ref 17) SEQ ID NO:37 Bt5 amino acid sequence (ref 17) SEQ ID NO:38 Bt5 polymorphic genomic sequence no. 1 SEQ ID NO:39 Bt5 polymorphic genomic sequence no. 2 SEQ ID NO:40 Bt5 polymorphic genomic sequence no. 3 SEQ ID NO:41 Bt5 polymorphic genomic sequence no. 4 SEQ ID NO:42 Bt5 polymorphic genomic sequence no. SEQ ID NO:43 Bt5 polymorphic genomic sequence no. 6 SEQ ID NO:44 Bt5 polymorphic genomic sequence no. 7 SEQ ID NO:45 Bt5 polymorphic genomic sequence no. 8 SEQ ID NO:46 Bt5 polymorphic genomic sequence no. 9 SEQ ID NO:47 Bt5 polymorphic genomic sequence no. SEQ ID NO:48 Bt5 polymorphic genomic sequence no. 11 SEQ ID NO:49 Bt5 polymorphic amino acid sequence nos. 1-3 and 7- SEQ ID NO:50 Bt5 polymorphic amino acid sequence no. 4 SEQ ID NO:51 Bt5 polymorphic amino acid sequence no. SEQ ID NO:52 Bt5 polymorphic amino acid sequence no. 6 SEQ ID NO:53 Bt5 polymorphic amino acid sequence no. 11 SEQ ID NO:54 Bt5 leader sequence (ref 17) SEQ ID NO:55 Bt5 cDNA variant no. 1(13) SEQ ID NO:56 Bt5 cDNA variant no. 2(7) SEQ ID NO:57 Bt5 cDNA variant no. 3(2) SEQ ID NO:58 Bt5 cDNA variant no. 4(15) SEQ ID NO:59 Bt5 cDNA variant no. 5(1) SEQ ID NO:60 Bt5 cDNA variant no. 6(11) SEQ ID NO:61 Bt5 cDNA variant no. 7(4) Received 12 December 2001 Pt j K F 'I.M k -13- SEQUENCE IDENTIFIER DESCRIPTION SEQ ID NO:62 Bt5 cDNA variant no. 8(12) SEQ ID NO:63 Bt5 cDNA variant no. 9(24) SEQ ID NO:64 Bt5 cDNA variant no. 10(9) SEQ ID NO:65 Bt5 cDNA variant no. 11(3) SEQ ID NO:66 Bt5 cDNA variant no. 12(5) SEQ ID NO:67 Bt5 cDNA variant no. 13(10) SEQ ID NO:68 Bt5 polymorphic amino acid variant no. 7 SEQ ID NO:69 Bt5 polymorphic amino acid variant no. 24 SEQ ID NO:70 Bt5 polymorphic amino acid variant no. 13 SEQ ID NO:71 Bt5 polymorphic amino acid variant no. SEQ ID NO:72 DNA primer SEQ ID NO:73 DNA primer SEQ ID NO:74 Nucleotide sequence of clone encoding BtA2 SEQ ID NO:75 Amino acid sequence encoded by SEQ ID NO:74 SEQ ID NO:76 Primer sequence SEQ ID NO:77 Primer sequence SEQ ID NO:78 Primer sequence SEQ ID NO:79 Primer sequence

LI

Received 12 December 2001 P?.OpaWE1kPas33rN~bm bdW*"l- -12/121I 14 TABLE 3 Variant Position Residue Hyrdophobic* Charge Chemical Genomic SEQ D NO:36,38-41 43-47 4 Lys Phi positive basic SEQ ID NO:42 4 Glu Phi negative acidic SEQ ID3 NO:36, 38-42 44 12 Asn Phi neutral amide SE47 O4 12 Asp Phi negative acidic SEQ ID NO:36, 38-40 42- 17 Leu Pho neutral aliphatic 17 Trp Pho neutral aromatic SEQ IDNO:41 SEQ lID NO:36, 38-41 43- 3 Leu Pho neutral aliphatic SE4D7 O4 33 Ile Pho neutral aliphatic SEQ ID NO:36, 38-42 44 34 Leu Pho neutral aliphatic 4734HsPipstv bai SEQ ID NO:4334HsPipstv bac SEQ ID NO:36, 38-42 44-. 117 Gln Phi neutral aliphatic SE4D7 O4 117 Lys Phi positive basic SEQ D NO:36, 38-47 2 Glu Phi negative acidic SEQ lBD NO:48 2 Gln Phi neutral aliphatic cDNA SEQ ID NO:36, 56 67 28 Lys Phi positive basic SEQ ID NO:63 28 Asn Phi neutral amide SEQ ID NO:36, 56 63 35 Tyr Pho neutral aromatic SEQ ID NO:67 35 Asp Phi negative acidic SEQ HD NO: 36, 63 67 57 Mrg Phi positive basic SEQ ID NO:56 57 Leu Pho neutral aliphatic Phi: hydrophilic and Pho: hydrophobic Received 12 December 2001 BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a diagrammatic representation showing the culture apparatus for dust and storage mites.

Figure 2 is a representation of the nucleotide sequence alignment of Clone 33 (Btll) and Df 11.

Figure 3 is a representation of the nucleotide sequence and the deduced amino acid sequence of the full-length Btll cDNA gene after the hybridization of clone 33 and RACE product. The full-length Bt1 cDNA gene is composed of 3111 nucleotide base pairs with a 2625 base pairs open reading frame from position 326-2950. An ATG (Met) sequence is found at position 326-328 while a TAA (stop) codon is found at position 2951- 2953. The full-length sequence shows a 325 nucleotide base pairs of untranslated region on the 5' end and a 158 nucleotide base pairs of untranslated region on the 3'end. The gene codes for an 875-amino acid protein with a methionine on the N-terminus.

Figure 4 shows the amino acid sequence alignment of Btl 1 with several paramyosin isolated from different invertebrates. CLUSTAL W Multiple Sequence Alignment program shows a significant homology ofBtl 1 to Df 11 and 3 other invertebrate paramyosins. Btl 1 amino acid sequence shows 90% sequence homology with the Dermatophagoides farinae paramyosin (Df 11). Moreover, the Btl 1 amino acid sequence exhibited 58% homology to the Drosophila melanogaster paramyosin, and 50% homology with Onchocerca volvulus paramyosin and 49% homology to Caenorhabditis elegans paramyosin. Results of Basic BLAST search using the BLASTP 2.0 program with the National Center for Biotechnology Information (NCBI) databank likewise revealed a significant homology of between Btll gene and other invertebrates paramyosins such as Dirofilaria immitis, Brugia malayi, Schistosoma mansoni, Taenia soleum and Schistosomajaponicum.

Received 12 December 2001 P. OIIpa2 pctuf3370lg.kx l I12DM I -16- Figure 5 is a photographic representation showing SDS-PAGE (10% w/v) analysis of recombinant Btll protein expressed in E. coli. The marker with different molecular weights in kD is shown. Lanes 1 and 2 are two concentrations ofBtll loaded.

Figure 6 is a photographic representation showing autoradiography of human IgE dot blot assay showing the different intensity of IgE reactivity of the representative sera (1 to 4).

Serum 1 is negative. Serum 2 is slightly positive and serum 3 is more reactive than serum 4 is positive. Each dot is loaded with the following: Btl 1 recombinant protein; whole B. tropicalis mite extract; and GST recombinant protein as a negative control. 2 Ag per dot is loaded for recombinant Btll and recombinant GST; 5 #g per dot is loaded for B.

tropicalis extract, respectively.

Figure 7 is a representation of nucleotide sequence and deduced amino acid sequence of Clone 1.2.

Figure 8 is a representation of the nucleotide and deduced amino acid sequence (in oneletter code) of the BtlO allergen. Nucleotide sequence residues are numbered at the end of each row. The stop codon is marked with a dash.

Figure 9 is a representation of the amino acid sequence alignment of group 10 from various mite species, i.e. Lepidoglyphus desructor (Lp 10), Dermatophagoidesfarinae (Df and Dermatophagoides pteronyssinus (Dp 10). There are two Dp 10 amino acid sequences in the Genbank: Dp 10(A) [submitted by Smith, W] and Dp 10(S) [23].

Figure 10 is a representation of IgE plaque immunoassay of Singapore and Taiwanese sera against Bt l-cDNA containing clone. Blots 1-13 were probed with mite-allergic individual sera, whereas blot 14 was non-atopic serum control. Sera for blots 1-9 and 10-13 are from Singapore and Taiwan, respectively.

A I W//VI A'J I Received 12 December 2001 r:OprEji.pcmsW2337lOttsi. **all liinm endddoic-12/IMI -17- Figure 11 is a photographic representation of SDS-PAGE showing purified recombinant fusion protein and thrombin-cleaved of GST-BtlO. Lane 1, protein molecular weight marker; lane 2, purified GST-Btl0; and lane 3, BtlO.

Figure 12 is a photographic representation of IgE immunoblotting of two atopic sera (blot A and B) and one non-atopic serum (blot C) against, lane 1, molecular weight marker; lane 2, Bt crude extract; lane 3, GST; lane 4, GST-Btl0.

Figure 13 shows the one-letter amino acid sequences deduced from the genomic sequences of the Bt5 variants as shown in Figure 21. Number on the top indicates the position of the amino residue.

Figure 14 shows the nucleotide sequences of Bt5 cDNA variants Number on the right indicates the nucleotide position. Sequences underlined are the start, stop codons and polyadenylated sites. Bt5 is the sequence published by Arruda et al. Figure 15 is a representation of the nucleotide sequence of BtA2 (SEQ ID NO:74). The numbers indicate the nucleotide positions. The nucleotide sequence of the clone is 507 bp in length. This includes a linker sequence ggccagag, a 286 bp 3' untranslated region with a poly-A tail and a 218 bp probable truncated coding region for the recombinant protein with a stop codon (TAA) at nucleotide residue 219-221.

Figure 16 is a representation of the nucleotide sequence and the deduced amino acid sequence of clone BtA2 (SEQ ID NO:74 and SEQ ID NO:75). The numbers indicate the nucleotide positions. The inferred amino acid sequence from nucleotides 9-218 indicated that this partial clone codes for a protein of 66 residues, with 8 cysteine residues in the molecules.

Figure 17 is a photographic representation of the plaque IgE immunoassay showing the IgE reactivity of 19 mite allergic serum samples and 1 non-allergic serum sample tested with the recombinant protein BtA2. Eight serum samples showed strong positive, 7 serum Received 12 December 2001 P:NopCj*lprHM37Un f ^.lemni-4p- ^-^lsm-i -m H llM I -18samples showed slightly positive and 4 serum samples showed negative IgE reactivity.

Panel 20 represents the negative control. Panel numbers 2, 3, 4, 5, 6, 7, 8, 9, 11, 15, 16 and 17 are positive. Panel number 1, 14, 18 and 20 are slightly positive whereas panel number 12, 13 and 19 are negative.

Figure 18 is a photographic representation of the SDS-PAGE (10% w/v) analysis of recombinant BtA2 protein expressed in E. coli (Gel A) and in Pichia pastoris (Gel The gels are stained with Coomassie brilliant blue R-250. The recombinant protein of clone BtA2 is approximately 7 kD. The GST-fusion protein of clone BtA2 is approximately kD. From 1 litre culture of E. coli, 7-10 mg of GST-clone BtA2 fusion protein could be obtained. The maximum yield of secreted BtA2 protein in yeast was one the fourth day after methanol induction. The yield by then was approximately 2.5-3.5 mg for 1 litre of yeast culture. Gel A shows the molecular weight marker lane, two lanes with two different concentrations of GST-fusion BtA2 protein purified by glutathione affinity chromatography and two lanes with different concentrations of elute (GST-BtA2 fusion protein which has been cleaved with thrombin and then purified by glutathione affinity chromatography. The elute is the 7.0 kD BtA2 recombinant protein). Gel B shows the molecular weight marker lane and the time kinetics of protein expression (day 1 to day 4) after the addition of 100% w/v methanol (to a final concentration of to the growth medium of yeast culture.

Figure 19 is a photographic representation of the autoradiography of dot blot assay using the ECL detection reagents. The dot blot shows the IgE reactivity of the various sera (A- Each dot is loaded with the following: 2 gg of BtA2 recombinant protein, obtained after thrombin digestion of the GST-BtA2 fusion protein; 2 pg of GST-BtA2 fusion protein; 2 ug of GST-Bt5 fusion protein; 2 gg of GST alone; and 2 ug of B.

tropicalis extract, respectively.

Figure 20 shows the sequence of the overlapping synthetic peptide and the peptide variants. The peptides were designed to be 16-amino acid residues in length, overlapping by 13 amino acid residues. Peptides 35-67 are Bt5 peptides designed on the basis of Received 12 December 2001 -19seuqence published by Arruda et al. Peptides 80-111 are the peptide variatns of These peptide variants contain naturally occurring mutations of Bt5 proteins.

Figure 21 shows the nucleotide sequences of Bt5 genomic DNA obtained by PCR amplification from the environmental Bt mites. Intron sequences are indicated in lower cases. Sequences underlined ar the start and stop codons. Sequences 1 to 5 and 6 to 11 representing Bt5 gene sequences obtained from Colombian and Singapore Bt mites, respectively. Reference to Bt5 sequences refers to the published cDNA sequence reported by Arruda et al. Number on the right indicates the nucleotide position of the DNA sequence.

Figure 22 shows the comparison of the deduced amino acid sequences for the Bt5 cDNA variants. Number on the right indicates the position of the amino acid residue.

Figure 23 shows the sequence of the Bt5 peptide variants that contain at least one human T-cell epitope. These epitopes are located in the regions where sequence polymorphisms are found. weak T cell response; moderate T cell response; strong T cell response; no T cell response. Polymorphic amino acid residues were denoted by single letter abbreviations for amino acids and the number indicated the residue position.

I/ Received 12 December 2001 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides a novel protein from the domestic dust or storage mite or a derivative, homologue, analogue, chemical equivalent or mimetic thereof which protein is capable of inducing or facilitating the induction of an immune response to the domestic dust or storage mite. This protein is conveniently in isolated or purified form. The identification of this protein, and its encoding nucleic acid sequence, now permits, inter alia, the use of these molecules as therapeutic and diagnostic agents.

Accordingly, one aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a dust mite or a derivative, homologue or mimetic of said protein allergen.

Reference to "mite" should be understood in its broadest sense and includes small arachnids of the order Acari. Preferred mites include mites from the family Glycyphagidae, such as the domestic mites B. tropicalis which are found in house dust and storage mites.

Accordingly, another aspect of the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is selected from Btl 1, Btl 0 and BtA2, each as herein defined, or polymorphic variants thereof and a polymorphic variant of Bt5 as herein defined.

Allergens from mites and in particular B. tropicalis are referred to herein as "Bt" allergens.

Reference to a protein "allergen" from a mite should be understood to mean that when introduced, in an effective amount, to sensitized individuals or individuals who are susceptible to sensitization, the protein will elicit, induce or otherwise facilitate an immune response. In this regard, the immune response may be a humoral and/or a cellular immune Received 12 December 2001 Poppts\f237nm l- IVI -21response. In a preferred embodiment, the immune response comprises a humoral response component and, most particularly, an IgE response. In one particular embodiment, the allergen is tropomyosin or a homologue or derivative thereof. A "Bt" allergen includes a polymorphic variant of a Bt allergen. Examples of polymorphic variants include the polymorphic variants of Bt5. The Bt5 allergen was disclosed by Arruda et al. Examples of specific Bt5 polymorphic variants are those set forth in SEQ ID NO:38 to SEQ ID NO:53 (genomic sequences), SEQ ID NO:59 to SEQ ID NO:67 (cDNA sequences) and SEQ ID NO:68 to SEQ ID NO:71 (amino acid sequences).

According to this embodiment, there is provided an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is a polymorphic variant of Bt5 having an amino acid sequence as set forth in any one of SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71 or a derivative, homologue or mimetic thereof or having at least about 45% similarity to an amino acid sequence set forth in any one of SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71.

More particularly, the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is a polymorphic variant of Bt5 wherein said nucleotide sequence is substantially as set forth in any one of SEQ ID NO:38 to SEQ ID NO:40 or SEQ ID NO:55 to SEQ ID NO:67 or a derivative, homologue or mimetic thereof or a nucleotide sequence having at least about 45% similarity to any one of SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71 or a nucleotice sequence capable of hybridizing to any one of SEQ ID NO:38 to SEQ ID NO:40 or SEQ ID to SEQ ID NO:67 or a complementary form thereof under low stringency conditions.

Reference to a "sensitized" individual should be understood as a reference to an individual who has been previously exposed to an allergen and upon subsequent exposure to the same Received 12 December 2001 P:<pg^Lcsa 3371O H. I w ,m ,-12/tJ9 -22allergen mounts an immune response which utilizes memory B and/or T cells. An individual who is "susceptible" to the sensitization is reference to an individual who, upon exposure to the allergen for the first time, will mount a primary immune response to the allergen. The allergen may comprise one or more epitopic regions to which a humoral immune response is directed. It may also, or alternatively, comprise one or more peptide regions to which a T cell response is directed upon processing and presentation of the protein by an antigen presenting cell.

It should also be understood that the allergen defined herein will not necessarily induce an immune response in all individuals who are exposed to it. It should also be understood that even within a group of individuals who are responsive to the allergen, these individuals may be responsive only to a certain range of dosages of the allergen. In this regard, the principles of low and high dose tolerance are relevant wherein introduction of an immunogen, such as an allergen, at very high or very low doses sometimes induces tolerance.

In one preferred embodiment, the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen wherein said protein allergen is Btl 1 having an amino acid sequence as set forth in any one of SEQ ID NO:2 to SEQ ID NO:14 or a derivative, homologue or mimetic thereof or having at least about 45% or greater similarity to an amino acid sequence set forth in in SEQ ID NO:2 to SEQ ID NO:14. This protein is referred to herein as "Btl The corresponding gene is 'Btll More particularly, the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is Btll wherein said nucleotide sequence is as substantially set forth in SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 13 or a derivative, homologue or mimetic thereof or capable of hybridizing to SEQ ID NO:1 Received 12 December 2001 P:pBW,%Lpctm2337mbLq-ntiwft=i Sxar fl#9qmnniDS rrnr12/IWI -23or SEQ ID NO:2 or SEQ ID NO:13 or a complementary form thereof under low stringency conditions.

In another preferred embodiment, the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is Btl0 having an amino acid sequence as substantially set forth in SEQ ID NO:29 or SEQ ID NO: 31 or a derivative, homologue or mimetic thereof having at least about 45% similarity to amino acid sequences set forth in SEQ ID NO:29 or SEQ ID NO:31.

More particularly, the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is Btl0 wherein said nucleotide sequence is as substantially set forth in SEQ ID NO:28 or SEQ ID NO: 30 or a nucleotide sequence having at least 45% similarity to SEQ ID NO:28 or SEQ ID NO:30 or a nucleotide sequence capable of hybridizing to SEQ ID NO:28 or SEQ ID NO:30 or a complementary form thereof under low stringency conditions. This protein is referred to herein as "BtlO". The corresponding gene is 'BtO0".

In another preferred embodiment, the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is BtA2 having an amino acid sequence as set forth in SEQ ID NO:75 or a derivative, homologue or mimetic thereof or having at least about 45% or greater similarity to an amino acid sequence set forth in SEQ ID More particularly, the present invention provides an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a Received 12 December 2001 -24sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic of said protein allergen, wherein said protein allergen is BtA2 wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:74 or a derivative, homologue or mimetic thereof or a nucleotide sequence having at least about 45% similarity to SEQ ID NO:74 or a nucleotide sequence capable of hybridizing to SEQ ID NO:74. This protein is referred to herein as "BtA2". The corresponding gene is "BtA2 The terms "protein" and "polypeptide" are used interchangeably herein to refer to a polymer of amino acid residues. For convenience, a "peptide" includes a "polypeptide".

The term "similarity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.

Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise a sequence only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A Received 12 December 2001 Pmq(pcwl mak L4 12rm/IMIarrrm~yl10 25 "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e.

resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. The terms "sequence similarity" and "sequence identity" as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-bynucleotide basis or an amino acid-by-amino acid basis over a window of comparison.

Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base A, T, C, G, I) or the identical amino acid residue Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.

Received 12 December 2001 -26- Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.

Generally, low stringency is at from about 25-30 0 C to about 42 0 C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. In one particular embodiment, low stringency is regarded as low stringency at 42 0 C. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out Tm 69.3 0.41 [Marmur and Doty However, the Tm of a duplex DNA decreases by 1 C with every increase of 1% in the number of mismatch base pairs [Bonner and Laskey Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 42 0 C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20 0 C to 65 0 C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65 0

C.

Another aspect of the present invention contemplates a nucleic acid molecule or derivative thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:1, or a derivative, homologue or mimetic thereof or capable of hybridizing to SEQ ID NO:1 or its complementary form under low stringency conditions. This nucleotide sequence corresponds to "Btll Preferably, the present invention contemplates a nucleic acid molecule encoding Bt11 or derivative thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13 or a derivative, homologue or mimetic thereof or r1/..iit-LJUUIVILL. I Received 12 December 2001 P: p prpm\2'n137U tr 1712J201_di-~ lI -27capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13 or their complementary forms under low stringency conditions and which encodes an amino acid sequence corresponding to an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:13 or a sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:2 or SEQ ID NO:13.

An example of a derivative is a polymorphic variant defined of Bt11 is by the nucleotide sequence set forth in SEQ ID NO:27 and with a corresponding amino acid sequence set forth in SEQ ID NO:27.

More particularly, the present invention contemplates a Bt11-encoding nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO: 1 or SEQ ID NO:2 or SEQ ID NO:13.

Another aspect of the present invention contemplates a genomic nucleic acid molecule or derivative thereof capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13 or their complementary forms, or a derivative or homologue or mimetic thereof, under low stringency conditions of 42°C.

Reference herein to Btl 1 and BtJ1 should be understood as a reference to all forms of Btl 1 and Bt11, respectively, including, for example, any peptide and cDNA isoforms which arise from alternative splicing of Btll mRNA or mutants or polymorphic variants of Btll or Btl 1. To the extent that it is not specified, reference herein to Btl 1 and Bt11 includes reference to derivatives, homologs, analogues, chemical equivalents and mimetics thereof.

In particular, the terms "Btl 1" and "Bt11 include reference to polymorphic variants such as but not limited to the variant defined by the nucleotide sequence SEQ ID NO:27 and its corresponding amino acid sequence SEQ ID NO:27.

Another aspect of the present invention contemplates a nucleic acid molecule or derivative thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:28 or SEQ ID NO:30, or a derivative, homologue or mimetic thereof or capable of hybridizing to Received 12 December 2001 P\opa~ftpafl2V3Ha372mO l c. mit lc12/22/0IMI -28- SEQ ID NO:28 or SEQ ID NO:30 or complementary forms thereof under low stringency conditions. This nucleic acid molecule is referred to herein as "BtlO".

Preferably, the present invention contemplates a BtlO-encoding nucleic acid molecule or derivative thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:28 or SEQ ID NO:30 or a derivative, homologue or mimetic thereof or capable of hybridizing to SEQ ID NO:28 or SEQ ID NO:30 or their complementary forms under low stringency conditions and which encodes an amino acid sequence corresponding to an amino acid sequence set forth in SEQ ID NO:28 or SEQ ID NO:30 or a sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:28 or SEQ ID NO:30 and wherein said amino acid sequence defines a polypeptide having tropomyosin activity.

More particularly, the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:28 or SEQ ID This nucleic acid molecule is referred to herein as "BtlO". Reference herein to "BtlO" includes both a cDNA molecule and a genomic gene as well as any polymorphic variants thereof.

Another aspect of the present invention contemplates a genomic nucleic acid molecule encoding BtlO or derivative thereof capable of hybridizing to SEQ ID NO:28 or SEQ ID or complementary forms thereof or a derivative or homologue or mimetic thereof, under low stringency conditions of 42 0 C. The genomic nucleic acid molecule preferably encodes a tropomyosin.

Reference herein to BtlO and BtlO should be understood as a reference to all forms of Btl0 and BtlO, respectively, including, for example, any peptide and cDNA isoforms which arise from alternative splicing of BtlO mRNA or mutants or polymorphic variants of BtlO or BtlO. To the extent that it is not specified, reference herein to BtlO and BtlO includes reference to derivatives, homologs, analogues, chemical equivalents and mimetics thereof.

PCFU/AUOU/U1227 %pe.mX,^m^Wa- Received 12 December 2001 -29- Still another aspect of the present invention contemplates a nucleic acid molecule or derivative thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:74, or a derivative, homologue or mimetic thereof or capable of hybridizing to SEQ ID NO:74 or its complmentary form under low stringency conditions. This nucleic acid molecule corresponds to "BtA2".

More particularly, the present invention contemplates a nucleic acid molecule or derivative thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:74 or a derivative, homologue or mimetic thereof or capable of hybridizing to SEQ ID NO:74 or its complementary form under low stringency conditions and which encodes an amino acid sequence corresponding to an amino acid sequence set forth in SEQ ID NO:75 or a sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID Even more particularly, the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:74.

Reference herein to BtA2 and BtA2 should be understood as a reference to all forms of BtA2 and BtA2, respectively, including, for example, any peptide and cDNA isoforms which arise from alternative splicing of BtA2 mRNA or mutants or polymorphic variants of BtA2 or BtA2. To the extent that it is not specified, reference herein to BtA2 and BtA2 includes reference to derivatives, homologues, analogues, chemical equivalents and mimetics thereof.

The protein and/or gene is preferably from B. tropicalis. However, a protein and/or gene may also be isolated from other species of mite such as other mites from the family Glycyphagidae. The protein and/or gene may also be isolated from any non-mite species such as other members of the order Acari. The protein and/or gene may also be isolated from any mite or non-mite species other than those comprising the order Acari.

Collectively, the allergens are referred to herein as "Bt" allergens. The corresponding Received 12 December 2001 genes are referred to a Accordingly, Bt includes Btl 1, Btl0, Bt5 and BtA2 and Bt includes Bt11. BtO0. Bt5 and BtA2.

Without limiting the present invention to any one theory or mode of action, a Bt protein is proposed to be reactive with human IgE present in the serum of patients who are allergic to house dust or storage mites. It is thereby thought that a protein comprises at least one epitopic region to which a humoral immune response is directed in individuals who are sensitized to, or susceptible to, sensitization to mites. A Bt protein is defined by the amino acid sequence or its encoding nucleotide sequence as set forth above (see also Table 3).

The nucleic acid molecule encoding a Bt is preferably a sequence of deoxyribonucleic acids such as a cDNA sequence or a genomic sequence. A genomic sequence may also comprise exons and introns. A genomic sequence may also include a promoter region or other regulatory regions.

Another aspect of the present invention contemplates a genomic nucleic acid molecule or derivative thereof capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13 or their complemetnary forms or a derivative or homologue or mimetic thereof, under low stringency conditions.

Reference herein to Bt and Bt should be understood as a reference to all forms of Bt and Bt, respectively, including, for example, any peptide and cDNA isoforms which arise from alternative splicing of Bt mRNA or mutants or polymorphic variants of Bt or Bt. To the extent that it is not specified, reference herein to Bt and Bt includes reference to derivatives, homologs, analogues, chemical equivalents and mimetics thereof.

Derivatives include fragments, parts, portions, mutants, and mimetics from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, epitopic regions of the Bt. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid Received 12 December 2001 P,.0p.%E ,,,PMds 337nW -lItiJ -31residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. An example of substitutional amino acid variants are conservative amino acid substitutions. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.

Additions to amino acid sequences including fusions with other peptides, polypeptides or proteins.

Homologs of the protein contemplated herein include, but are not limited to, proteins derived from different species.

Chemical and functional equivalents of Bt or Bt should be understood as molecules exhibiting any one or more of the functional activities of Bt or Bt and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.

The derivatives of Bt include fragments having particular epitopes of parts of the entire Bt protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.

Analogues of Bt contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues.

Derivatives of nucleic acid sequences may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid Received 12 December 2001 np-ypm.NrpanWlnPW 3 l -A-QLitioSdd A M I -32molecules. The derivatives of the nucleic acid molecules of the present invention include nligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules. Derivatives of nucleic acid sequences also include degenerate variants.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with Nbromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide Received 12 December 2001 r^pu0|kpculm3 .k 121mnit iH/IMI -33or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, omithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid contemplated herein is shown in International Patent Application No. PCT/AU96/00668 [International Patent Publication No. WO 97/15663].

Crosslinkers can be used, for example, to stabilize 3D conformations, using homobifunctional crosslinkers such as the bifunctional imido esters having (CH 2 )n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.

The nucleic acid molecule of the present invention is preferably in isolated form or ligated to a vector, such as an expression vector. By "isolated" is meant a nucleic acid molecule having undergone at least one purification step and this is conveniently defined, for example, by a composition comprising at least about 10% subject nucleic acid molecule, preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 40-50%, even still more preferably at least about 60-70%, yet even still more preferably 80-90% or greater of subject nucleic acid molecule relative to other components as determined by molecular weight, encoding activity, nucleotide sequence, base composition or other convenient means. The nucleic acid molecule of the present invention may also be considered, in a preferred embodiment, to be biologically pure.

Received 12 December 2001 -34- As stated above; the term "protein" should be understood to encompass peptides, polypeptides and proteins. The protein may be glycosylated or unglycosylated and/or may contain a range of other molecules fused, linked, bound or otherwise associated to the protein such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins. Reference hereinafter to a "protein" includes a protein comprising a sequence of amino acids as well as a protein associated with other molecules such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.

In a particularly preferred embodiment, the nucleotide sequence corresponding to Btll is a cDNA or genomic sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13 or a derivative thereof including a nucleotide sequence having at least about 45% similarity to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13.

In another particularly preferred embodiment, the nucleotide sequence corresponding to BtlO is a cDNA or genomic sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:28 or SEQ ID NO:30 or a derivative thereof including a nucleotide sequence having at least about 45% similarity to SEQ ID NO:28 or SEQ ID In another particularly preferred embodiment, the nucleotide sequence corresponding to BtA2 is a cDNA or genomic sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:74 or d derivative thereof thereof including a nucleotide sequence having at least about 45% similarity to SEQ ID NO:74.

A derivative of a nucleic acid molecule of the present invention also includes a nucleic acid molecule capable of hybridizing to a nucleotide sequence as set forth in one or more of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13 or SEQ ID NO:28 or SEQ ID or any one of SEQ ID NO: 38 to SEQ ID NO: 48 or SEQ ID NO:55 to SEQ ID NO:67 or SEQ ID NO:74 or complementary forms thereof under low stringency conditions.

Preferably, low stringency is at 42 0

C.

Received 12 December 2001 p: ~paEih.pc y37ro bi.hiaanM.c dp im 4 .c-H /IIII ZVI The nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell E.coli) or a eukaryotic cell yeast cells, fungal cells, insect cells, mammalian cells or plant cells). The nucleic acid molecule may be ligated or fused or otherwise associated with a nucleic acid molecule encoding another entity such as, for example, a signal peptide. It may also comprise additional nucleotide sequence information fused, linked or otherwise associated with it either at the 3' or 5' terminal portions or at both the 3' and 5' terminal portions. The nucleic acid molecule may also be part of a vector, such as an expression vector. The latter embodiment facilitates production of recombinant forms of Bt which forms are encompassed by the present invention.

The present invention extends to the expression product of the nucleic acid molecules as hereinbefore defined.

One expression product is Btl 1 having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:14 or is a derivative, homologue, analogue, chemical equivalent or mimetic thereof as defined above or is a derivative, homologue or mimetic having an amino acid sequence of at least about 45% similarity to at least 10 contiguous amino acids in the amino acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:14 or a derivative or homologue or mimetic thereof.

Accordingly, another aspect of the present invention is directed to an isolated protein selected from the list consisting of:a protein allergen from a mite or a derivative, homologue, analogue, chemical equivalent or mimetic thereof; (ii) a protein allergen from B. tropicalis or a derivative, homologue, analogue, chemical equivalent or mimetic thereof; Received 12 December 2001 OpaEsX pat37OLbLc a a .imnf d doc-12/1Mt -36- (iii) a protein having an amino acid sequence substantially as set forth in SEQ ID NO:2 or SFQ ID NO:14 or a derivative, homologue or mimetic thereof or a sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:2 or SEQ ID NO:14 or a derivative, homologue, analogue, chemical equivalent or mimetic of said protein; (iv) a protein encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:14 or a derivative or homologue thereof or a sequence encoding an amino acid sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:2 or SEQ ID NO:14 or a derivative, homologue, analogue, chemical equivalent of said protein; a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO: 14 or complementary forms thereof or a derivative or homologue thereof under low stringency conditions and which encodes an amino acid sequence substantially as set forther in SEQ ID NO:2 or SEQ ID NO:14 or a derivative or homologue or mimetic thereof or an amino acid sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:2 or SEQ ID NO:14; (vi) a protein as defined in paragraphs or (ii) or (iii) or (iv) or in a homodimeric form; and (vii) a protein as defined in paragraphs or (ii) or (iii) or (iv) or in a heterodimeric form.

Yet another expression product is Btl0 having tropomyosin activity and an amino acid sequence set forth in SEQ ID NO:29 or is a derivative, homologue, analogue, chemical equivalent or mimetic thereof as defined above or is a derivative, homologue or mimetic having an amino acid sequence of at least about 45% similarity to at least 10 contiguous Received 12 December 2001 rP~alEpacs~fl27 ba I ti- i nrr H m..IM. I -37amino acids in the amino acid sequence as set forth in SEQ ID NO:29 or a derivative or homologue or mimetic thereof.

Accordingly, another aspect of the present invention is directed to an isolated protein selected from the list consisting of:a protein allergen from a mite or a derivative, homologue, analogue, chemical equivalent or mimetic thereof; (ii) a protein allergen from B. tropicalis or a derivative, homologue, analogue, chemical equivalent or mimetic thereof; (iii) a protein having an amino acid sequence substantially as set forth in SEQ ID NO:29 or SEQ ID NO:31 or a derivative, homologue or mimetic thereof or a sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:29 or SEQ ID NO:31 or a derivative, homologue, analogue, chemical equivalent or mimetic of said protein; (iv) a protein encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:28 or SEQ ID NO:30 or a derivative or homologue thereof or a sequence encoding an amino acid sequence having at least about 45% similarity to at least contiguous amino acids in SEQ ID NO:28 or SEQ ID NO:30 or a derivative, homologue, analogue, chemical equivalent of said protein; a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:28 or SEQ ID NO:30 or complementary forms thereof or a derivative or homologue thereof under low stringency conditions and which encodes an amino acid sequence substantially as set forth in SEQ ID NO:28 or SEQ ID NO:30 or a derivative or homologue or mimetic thereof or an amino acid sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:28 or SEQ ID Received 12 December 2001 -38- (vi) a protein as defined in paragraphs or (ii) or (iii) or (iv) or in a homodimeric form; and (vii) a protein as defined in paragraphs or (ii) or (iii) or (iv) or in a heterodimeric form.

Even still yet another expression product is BtA2 having an amino acid sequence set forth in SEQ ID NO:75 or is a derivative, homologue, analogue, chemical equivalent or mimetic thereof as defined above or is a derivative, homologue or mimetic having an amino acid sequence of at least about 45% similarity to at least 10 contiguous amino acids in the amino acid sequence as set forth in SEQ ID NO:75 or a derivative or homologue or mimetic thereof.

Accordingly, another aspect of the present invention is directed to an isolated protein selected from the list consisting of:a protein allergen from a mite or a derivative, homologue, analogue, chemical equivalent or mimetic thereof; (ii) a protein allergen from B. tropicalis or a derivative, homologue, analogue, chemical equivalent or mimetic thereof; (iii) a protein having an amino acid sequence substantially as set forth in SEQ ID NO:75 or a derivative, homologue or mimetic thereof or a sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID NO:75 or a derivative, homologue, analogue, chemical equivalent or mimetic of said protein; (iv) a protein encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:74 or a derivative or homologue thereof or a sequence encoding an amino acid sequence having at least about 45% similarity to at least 10 contiguous amino acids Received 12 December 2001 P:.OpulEj.pa 1 03372 tl p ic.l.. im 4 1.11i1 39 in SEQ ID NO:75 or a derivative, homologue, analogue or chemical equivalent of said nrotein; a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:74 or a complementary form thereof or a derivative or homologue thereof under low stringency conditions and which encodes an amino acid sequence substantially as set forth in SEQ ID or a derivative or homologue or mimetic thereof or an amino acid sequence having at least about 45% similarity to at least 10 contiguous amino acids in SEQ ID (vi) a protein as defined in paragraphs or (ii) or (iii) or (iv) or in a homodimeric form; and (vii) a protein a defined in paragraphs or (ii) or (iii) or (iv) or in a heterodimer form.

The present invention further extends to fragments of the Bt allergens comprising a linear or conformational epitope. For example, preferred fragments of Bt5 are set forth in Figure The Bt of the present invention may be in multimeric form meaning that two or more molecules are associated together. Where the same Bt molecules are associated together, the complex is a homomultimer. An example of a homomultimer is a homodimer. Where at least one Bt is associated with at least one non-Bt molecule, and the complex is a heteromultimer such as a heterodimer. The heteromultimer may include, for example, another molecule in an amount capable of inducing tolerance to an allergen.

The ability to produce recombinant Bt permits the large scale production of Bt for commercial use. The Bt may need to be produced as part of a large peptide, polypeptide or protein which may be used as is or may first need to be processed in order to remove the Received 12 December 2001 extraneous proteinaceous sequences. Such processing includes digestion with proteases, peptidases and amidases or a range of chemical, electrochemical, sonic or mechanical disruption techniques.

Notwithstanding that the present invention encompasses recombinant proteins, chemically synthetic techniques also preferred in synthesis of Bt.

Bt according to the present invention is conveniently synthesized based on molecules isolated from B. tropicalis. Isolation of the B. tropicalis molecules may be accomplished by any suitable means such as by chromotographic separation, for example using CMcellulose ion exchange chromotography followed by Sephadex G-50 column) filtration. Many other techniques are available including HPLC, PAGE amongst others.

Once purified, the Bt molecule can be partially sequenced and/or fragments produced induced directly as a source of Bt or at a template for amino acid synthesis.

Bt may be synthesized by solid phase synthesis using F-moc chemistry. Bt and fragments thereof may also be synthesized by alternative chemistries including, but not limited to, t- Boc chemistry or by classical methods of liquid phase peptide synthesis.

In accordance with the present invention, it is proposed that Bt is a mite derived protein allergen from mites which comprises at least one epitopic region to which an individual sensitized to mites, or an individual who is susceptible to sensitization to mites, may mount an immune response, such as a humoral IgE response. The identification of a novel mite allergen permits the generation of a range of molecules, such as therapeutic and prophylactic molecules, for the treatment of conditions such as mite induced allergies. The identification of Bt also facilitates the generation of molecules for use as diagnostic agents.

Accordingly, the present invention provides a method of preventing, reducing or otherwise ameliorating a Bt hypersensitivity condition in a subject said method comprising administering to said subject an effective amount of Bt or a derivative, homologue, Received 12 December 2001 PXopnr~prLP~ntM37Um; l dw- 121 12YMI -41analogue, mimetic or chemical equivalent thereof for a time and under conditions sufficient to desensitize said individual.

The individual who is treated in accordance with the method of the present invention may be human or animal in need of therapeutic or prophylactic treatment and includes an individual who has become sensitized, or who is predisposed to becoming sensitized, to at least part of the Bt molecule such as an epitopic region of Bt. The Bt molecule, or part thereof, to which an individual becomes sensitized, may comprise part of any antigen such as, but not limited to, the dust or storage mite or a non-mite species.

Reference to "subject" should be understood as a reference to all animals including primates humans, monkeys), livestock animals sheep, cows, horses, donkeys, goats, pigs laboratory tests animals rats, guinea pigs, rabbits, hamsters), companion animals dogs, cats), captive wild animals emus, kangaroos, deer, foxes) avies chickens, ducks, bantoms, pheasants, emus, ostriches), reptiles lizards, snakes, frogs) and fish trout, salmon).

Reference to "Bt hypersensitivity condition" should be understood as a reference to any of type I, II, III or IV hypersensitivity conditions directed to all or part of Bt. More particularly, the Bt hypersensitivity condition is a type I hypersensitivity condition.

Examples of type I hypersensitivity conditions which may be treated in accordance with the method of the present invention include, but are not limited, atopy, immediate hypersensitivity, systemic anaphylaxis, allergic rhinitis (hayfever) or asthma (for example bronchial asthma).

Although the preferred method is to reduce or prevent the induction of an immune response to an antigen comprising all or part of Bt, it may be desirable to induce or upregulate an immune response to such an antigen where, for example, the antigen is not innocuous. For example, where the antigen is a bacterium or parasite which comprises a all or part of a Bt region, it would be desirable to up-regulate an immune response to Bt.

Aeceived 12 December 2001 42 Accordingly, another aspect of the present invention relates to a method of modulating, in a subject, an immune response directed to Bt, said method comprising administering to said subject an effective amount of Bt or a derivative, homologue, analogue, chemical equivalent or mimetic thereof for a time and under conditions sufficient to up-regulate, down-regulate or otherwise modulate said immune response.

Reference to an "effective amount" should be understood as a reference to an amount of Bt or derivative, homologue, analogue, chemical equivalent or mimetic thereof necessary to at least partly achieve the desired outcome. For example, where it is sought to induce tolerance to an antigen comprising a Bt portion, very low or very high concentrations of Bt may be administered to induce low or high dose tolerance, respectively. Alternatively, where it is sought to induce an immune response, doses of Bt which do not induce tolerance may be administered.

The present invention further extends to the use of Bt in the manufacture of a medicament for modulating an immune response.

Yet another aspect of the present invention provides an agent useful for modulating an immune response, said agent comprising Bt as hereinbefore defined.

Preferably, said modulation is down regulation of the immune response.

In accordance with these methods, more than one type of protein or peptide may be administered. For examnple, where the Bt is administered for the purpose of inducing tolerance, the Bt may be co-administered with other known tolerance inducing compounds or molecules. Alternatively, where the Bt is administered to up-regulate the immune response, the Bt may be administered with an adjuvant. By "co-administered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By "sequential administration" is meant a time difference of from seconds, minutes, hours or Aeceived 12 December 2001 -43days between the administration of the two or more types of molecules. The Bt and other comnnlond or mnlec-iul may he administered in any order.

Routes of administration include but are not limited to intravenously, intraperitoneal, subcutaneously, intracranial, intradermal, intramuscular, intraocular, intrathecal, intracerebrally, intranasally, infusion, orally, rectally, via iv drip, patch and implant.

Intravenous routes are particularly preferred. Administration may also be via aerosol or inhalation.

Another aspect of the present invention provides a composition for use in modulating an immune response comprising Bt as hereinbefore defined and one or more pharmaceutically acceptable carriers and/or diluents. The composition may also comprise two different types of molecules such as Bt and another compound or molecule with which it is coadministered.

Compositions suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. They must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients Received 12 December 2001 1 E.OW UCW7O=hL bi WK"ifli1 2112MI -44enumerated above, as required, followed by, for example, filter sterilization or sterilization hby Othr apprnnopriate means. Dispersions are also contemplated and these may be prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, a preferred method of preparation includes vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution.

When the active ingredients are suitably protected, they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ng and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter. A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or teceived 12 December 2001 elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.

Any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

The present invention also extends to forms suitable for topical application such as creams, lotions and gels.

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active material and the particular therapeutic effect to be achieved and the limitations inherent in the art of compounding such an active material.

Effective amounts of protein contemplated by the present invention will vary depending on the severity of the pain and the health and age of the recipient. In general terms, effective amounts may vary from 0.01 ng/kg body weight to about 100 mg/kg body weight.

Aeceived 12 December 2001 -46- Alternative amounts include for about 0.1 ng/kg body weight about 100 mg/kg body weight or from 1.0 ne/kg body weight to about 80 mg/kg body weight.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of expressing Btl 1 or derivative, homologue or mimetic thereof.

In particular, the composition comprises an isolated nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from a mite or a derivative, homologue or mimetic or said protein allergen.

Preferably, the nucleotide sequence encodes the amino acid sequence of Btl 1 substantially as set forth in SEQ ID NO:2 or SEQ ID NO:14 or a derivative, homologue or mimetic thereof or having at least about 45% or greater similarity to at least 10 contiguous amino acids in SEQ ID NO:2 or SEQ ID NO:14.

Even more preferably, the nucleotide sequence comprises the Btl1 nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13, or a derivative, homologue or mimetic thereof, or capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13 under low stringency conditions.

Preferably, the nucleotide sequence encodes the amino acid sequence of Btl0 substantially as set forth in SEQ ID NO:29 or SEQ ID NO:31 or a derivative, homologue or mimetic thereof or having at least about 45% or greater similarity to at least 10 contiguous amino acids in SEQ ID NO:29 or SEQ ID NO:31 and which corresponds to a polypeptide having tropomyosin activity.

Even more preferably, the nucleotide sequence comprises the BtlO nucleotide sequence of SEQ ID NO:28 or SEQ ID NO:30 or a derivative, homologue or mimetic thereof, or capable of hybridizing to SEQ ID NO:28 or SEQ ID NO:30 under low stringency conditions.

/.?.17i7 I.

Received 12 December 2001 F'XpWJpaOc22O37=.k=Nnrpp- 'Wing d"-t -47- Preferahly, the nucleotide sequence encodes the amino acid sequence of BtA2 substantially as set forth in SEQ ID NO:75 or a derivative, homologue or mimetic thereof or having at least about 45% or greater similarity to at least 10 contiguous amino acids in SEQ ID Even more preferably, the nucleotide sequence comprises the BtA2 nucleotide sequence of SEQ ID NO:74, or a derivative, homologue or mimetic thereof, or capable of hybridizing to SEQ ID NO:74 under low stringency conditions.

The present invention further contemplates the introduction of naked DNA as well as the viral or bacterial delivery of nucleic acid molecules.

Still another aspect of the present invention contemplates a method for the prophylactic treatment of an allergic condition comprising the step of administering to an individual nucleic acid molecule comprising a gene encoding Bt or a derivative or homologue thereof whereby airway hyper-reactivity or airway inflammation is prevented.

Preferably, the allergic condition is allergic asthma, atophic dermatitis and/or rhinitis.

More preferably, the nucleic acid molecule is in the form of an eukaryotic expression vector. The eukaryotic expression vector may be selected from the group consisting of vectors with CMV promoter, RSV promoter and SV40 promoter and is preferably pCMV.

The allergen may include any environmental antigen which can induce allergic reaction in human such as mite allergens, glutathione S-transferase, pollen, animal dander, house dust and peanut and the like.

The preferred allergic diseases include, for example, allergic asthma, allergic rhinitis, atopic dermatitis and anaphylaxis.

Received 12 December 2001 I'op FL pB \237 0 l7,Il. cc" C «rr n tald-1r2//I01 -48- Preferably, the eukaryotic expression vector is administered in a pharmaceutical composition comprising a carrier selected from the group consisting of normal saline and a liposome. The pharmaceutical compositions of the invention are preferably administered by intramuscular injection, intranasal delivery or intratracheal delivery. The pharmaceutically acceptable carrier may be conventional carriers useful for intramuscular injection, intranasal delivery or intratracheal delivery known in the art. For example, a physiologically acceptable buffer solution, normal saline, gold bead or liposome may be used.

Depending on the characteristic and progression of the disease to be prevented or treated in the individual and other factors such as age and physical conditions of the patient, the dosage of the recombinant plasmids ranges from about 0.01 to about 1.0 mg/kg body weight for a patient treated in accordance with the present invention.

Preferably, the individual is a human.

In relation to allergen gene transfer, this is predicated on DNA-based immunization which induces a biased Thl immune response. This offers a strategy for modulating Th2 associated responses. In a preferred embodiment, allergen gene transfer immunization is by the intramuscular injection of a plasmid DNA encoding a house dust or storage mite allergen (28).

Yet another aspect of the present invention is directed to antibodies to Bt and their derivatives, homologues, analogues, mimetics and chemical equivalents thereof. Such antibodies may be monoclonal or polyclonal.

In the case of small peptides, these may first need to be associated with a carrier molecule.

The antibodies of the present invention are particularly useful as therapeutic or diagnostic agents. For example, specific antibodies can be used to screen for Bt immunoassays or used as antagonists to inhibit Bt activity under certain circumstances such as where temporary hypersensitivity inhibition only is required. Techniques for such immunoassays Received 12 December 2001 -49are well known in the art and include, for example, sandwich assays and ELISA.

Knowledge of Bt levels may be important for monitoring certain therapeutic protocols.

Antibodies to the Bt (or its derivatives, homologues, analogues or mimetics) of the present invention may be monoclonal or polyclonal. Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies.

As stated above, specific antibodies can be used to screen for the Bt. The latter would be important, for example, as a means for screening for levels of Bt in a biological fluid or purifying Bt made by recombinant means from culture supernatant fluid.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available antiimmunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of Bt.

Both polyclonal and monoclonal antibodies are obtainable by immunization with Bt and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of Bt or antigenic parts thereof, collecting serum from the animal and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The Received 12 December 2001 P-o=.c u-7l7)OL w preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and vlymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.

Even still another aspect of the present invention is directed to methods of diagnosing individuals who have become sensitized to antigens which comprise all or part of Bt.

Accordingly, another aspect of the present invention contemplates a method for detecting antibody directed to all or part of Bt in a biological sample from a subject, said method comprising contacting said biological sample with Bt or a derivative, homologue, analogue, chemical equivalent or mimetic thereof for a time and under conditions sufficient for an antibody-protein complex to form, and then detecting said complex.

Detection of the presence of Bt (for example, in a dust sample) or antibody to Bt may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S.

Patent Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target, such as Bt.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention to detect Bt or antibody to Bt. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody of Bt is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen (or Bt-antibody) complex, a second antibody specific to the complex molecules, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed Received 12 December 2001 O PaM233710t *a gIV I- I2)I -51 away, and the presence of the antigen or antibody is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the sample is one which might contain an antibody to Bt including cell extract, culture supernatant tissue biopsy, serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture. However, the sample may also be a sample thought to comprise the Bt molecule, such as a sample of dust thought to comprise dust or storage mite.

In the typical forward sandwich assay, a first antibody having specificity for the protein or antigenic parts thereof is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient 2-40 minutes or overnight if more convenient) and under suitable conditions from room temperature to about 37°C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

Received 12 December 2001 52 An alternative method involves immobilizing the target molecules (such as Bt) and then exposing the immobilized target to a sample which is to be tested for the presence of antibody to Bt. A second labelled reporter antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, luciferase glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody-peptide complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibodyantigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

I Received 12 December 20b 1 -53- Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope.

As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibodyhapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method.

However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

Further features of the present invention are more fully described in the following nonlimiting Examples.

Received 12 December 2001 U.2337«LMh<«lcll *I2IIZ/U7 -54- EXAMPLE 1 Culturing and harvesting of B. tropicalis mites Bt dust or storage mites were grown in the laboratory and the starter cultures were prepared by collecting mites from the house dust samples in Singapore. Bt were identified according to Bronswijk (18) and isolated under a stereomicroscope from the dust samples, which were used for setting-up a starter culture (Figure Fine powdered tetramin fish feed was used as a culture medium for culturing Bt. The tetramin flakes were heated at 60 0 C for 3 hours before being grounded to kill pre-existing mites and insects. It was then sieved through a 125 pm sieve before use. In order to provide a maximum surface area, a thin layer of <125 pm particles were introduced into Erlenmeyer flasks together with a few flakes that served as shelters and breeding ground for mites. A starter culture from a small bottle was inoculated into a one-litre Erlenmeyer flask which was then covered by two layers of paper towels to allow ventilation, and sealed with masking tape around the opening of the flask to prevent contamination from the other mites and insects. Those cultures were grown under natural environmental conditions with a mean annual temperature of 30 0 C and a mean RH of Harvesting of dust or storage mites was performed when the culture was approximately 4weeks old. Culture was observed under stereomicroscope to determine the purity of the mite populations. The mite culture was separated through a series of 500 pm and 125 pm sieves, by using a mechanical sieve shaker, where vibration was applied for about minutes. Mites with sizes greater than 120 pm were transferred to a modified Tullgren, which was built up of 5 layers of gauze on a funnel that attached to a 15ml-Falcon tube (2097). A 60W bulb was applied from a distance of 15 cm from the culture medium for 4 hours. Most of the mites in the medium crawled through the gauze and down into the tube.

Mites that remained in the funnel would then swept into the tube by a tiny soft brush after the gauze was removed. The purified mites were stored -80°C until use. Mites and the powered medium particles that were finer than 125 pm collected in a receiving pan placed at the bottom of the sieves were used for further subculturing.

Aeceived 12 December 2001 rp:N pci po\237W 0l.bL.om cil -12/12/01 "Examples 2 to 13 relate to Btll EXAMPLE 2 Screening ofBt cDNA Library B. tropicalis cDNA library (Uni-ZAP XR Library) was screened by colony/plaque hybridization using the DIG system filter hybridization (Boehringer Mannheim).

Digoxigenin (DIG)-labelled fragment 2 (f2) and fragment 4 (f4) of Dfl 1 with nucleotides lengths of 431 and 604 bp, respectively, were used as molecular probes in the screening process. Phages were plated at 5-10,000 plaque forming units (pfu's) per 145 mm NZY agar petri dish. Pre-cooled plaques with a diameter of 1-2 mm after overnight incubation at 37 0 C were transferred to Hybond-N+ Nylon transfer membranes (Amersham) by colony/plaque lift method. Membranes were soaked in denaturation solution (0.5 N NaOH and 1.5 NaC1) for 5 minutes; followed by neutralization solution (1.0 M Tris-HCl and M NaCI, pH 7.5) for 15 minutes; and in 2X SSC solution (0.3 mM NaCI and 30 mM sodium citrate, pH Transferred DNA was crosslinked into the membrane by using a UV Stratalinker 1800 (Stratagene) at 1,200 gJoules x 100. Hybridization with the labelled probes was done overnight at 42.5 0 C in a hybridization chamber (Maxi 14 HybaidT).

Membranes were washed twice for 10 minutes each with 2X SSC (with 0.1% w/v SDS) at room temperature followed by two more washes at 15 minutes each with 0.5% SSC (with 0.1% w/v SDS) at 65 0 C. Unspecific sites were blocked with 10% blocking solution (Boeringer Mannheim) for 1 hour prior to incubation with anti-DIG antibody (1:10,000) for 30 minutes. After 4 times of washing with wash buffer (0.1 M Maleic acid, 0.15 M NaCI, 0.3% v/v Tween 20) for 15 minutes each, membranes were incubated for 2 minutes in detection buffer (0.1 Tris-HCl, 0.1 NaCI). Membranes were finally incubated in CSPD solution (1:100) for 5 minutes. Membranes placed in saran wrap were exposed to BioMax film (Kodak) for 15 minutes and developed in a dark room. Positive pfu's were picked from the agar plate using a cut 200 il pipet tip. Clones were incubated at least for 24 hours in SM medium in a rotator drive. Clones were purified further by secondary and tertiary screening.

Aeceived 12 December 2001 -56- EXAMPLE 3 Transformation of Positive Phage Clones Purified clones were transformed into E. coli SOLR strain using the single-clone excision protocol described in the Uni-ZAP XR Library Instruction Manual (Stratagene). Briefly, a mixture of 200 l of E. coli XL1-Blue 30 pl of positive phage stock clone (IX) and 1 gl of ExAssist helper phage x 10 6 pfu/gl) was incubated at 37 0 C for minutes. After the addition of 3 ml of LB broth, the reaction mixture was incubated for 3 hours at 37 0 C in a shaker (250 rpm). The cells were then heated at 70 0 C for 20 minutes in a water bath followed by centrifugation at 3,500 rpm for 15 minutes. Two microliters of the supernatant was added to 150 ll of overnight culture of E.coli SOLR strain The reaction culture was incubated for 15 minutes at 37 0 C before plating 100 ll of the cell mixture on LB-ampicillin agar plates. Plates were incubated overnight at 37 0

C.

Transformed clones were subcultured overnight in LB-ampicillin medium. Glycerol stock of the different clones were stored at -80 0 C for future use.

EXAMPLE 4 DNA Sequencing Phage DNA of positive clones isolated and purified by using the Wizard Plus SV minipreps DNA purification system (Promega) were determined and analyzed by DNA sequencing. 50 to 80 ng of the amplified DNA was added to a 20 gl reaction mixture containing 10 pmol of either T3 primer (5'AATTAACCCTCACTAAAGGG3') (SEQ ID NO:19) or T7 primer (3'CGGGATATCACTC-AGCATAATG5') (SEQ ID NO:20) and 8 pl of Big Dye terminator RR mix (PE Applied Biosystems). Amplification was performed by 25 cycles using the PTC-100 [Trademark] Programmable Thermal Controller (MJ Research Inc., USA). Each cycle consists of 96°C for 30 seconds, 50 0 C for 15 seconds and 0 C for 4 minutes. DNA sequencing was performed using the ABI 377 DNA Sequencer (Applied Biosystems).

Received 12 December 2001 P'O.pT\r Up 370S.bLe 7 mb* p^lp* ,lhri -,fT in 12/12M/H -57- EXAMPLE Sequence Analysis The Btl nucleotide sequence was compared and aligned with the Df 11 nucleotide sequence using the CLUSTAL W version 1.6 multiple sequence alignment software. The reverse and complement sequences were performed using the GCG sequence analysis package. Likewise, the deduced nucleotide sequence was submitted to the databases of the National Center for Biotechnology Information (NCBI), using the BLAST network server for nucleotide and amino acid homology searches.

EXAMPLE 6 Human IgE Reactivity ofBtll clone An initial experiment by plaque immunoassay to determine the frequency of human IgE reactivity ofBtll clone 2 was carried out with 23 sera from donors with positive skin prick test to B. tropicalis extract. Prior to the experiment, human sera were adsorbed overnight at 4°C with XL1-Blue E. coli lysate. t 11 clone 2 was plated at approximately 1,000 plaqueforming units (pfu's) per 145 mm NZY agar plate. Plaques were transferred to nitrocellulose membrane [Hyband-C Extra membrane that was saturated with isopropyl-3- D-thiogalactopyranoside (IPTG)] by plaque lift method. After blocking the membrane with solution of non-fat milk powder for 1 hour, it was incubated overnight with human sera (in 1% non-fat milk) at 4 0 C, followed by one hour incubation with alkaline phosphatase conjugated monoclonal anti-human IgE (1:10,000 dilution) at room temperature. Three times washing with PBS containing 0.05% v/v Tween 20 (PBST) was done in between the incubation steps. Membranes blotted on paper towels were finally incubated for 15-30 minutes in a developing solution containing 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) and nitro blue tetrazolium (NBT, Sigma) in 10 ml of AP buffer An intense bluish purple colour reaction indicated a positive reaction.

Received 12 December 2001 P:I)ai^pdl2337«U 'o,aiM ai -58- EXAMPLE 7 Amplification of cDNA Ends The 5'end of Btll cDNA clone was isolated using the 5'-Rapid Amplification of cDNA Ends (5'-RACE) following the protocol described in the SMART [Trademark] RACE cDNA Amplification Kit User Manual, protocol number PT3269-1 version PR93377 (Clontech Laboratories, Inc.) with slight modifications. Briefly, first-strand cDNA synthesis was performed using 1 ljg of total RNA isolated from B. tropicalis culture. RACE Ready cDNA reaction mixture composed of 1 pl total RNA sample (1 ig), 1 l cDNA Synthesis (CDS) primer, 1 Il SMART II oligo, and 2 ul RNAse-free water was incubated at 70 0 C for 2 minutes followed by cooling on ice for 2 minutes. The 5 tlreaction mixture was then mixed with 2 jil 5 x first-strand buffer, 1 ll 20 mM DTT, 1 Il mM dNTP mix and 1 Il Superscript II (200 units/pl, Gibco BRL, USA). The reaction mixture was then incubated at 42 0 C for 90 minutes in an air incubator prior to dilution with 100 ul of Tricine-EDTA Buffer. The diluted reaction product was then heated at 72 C for 7 minutes. 5'-Rapid Amplification of cDNA Ends (5'-RACE) was performed using a 50 ul PCR reaction mixture composed of 2.5 tl 5'-RACE-Ready cDNA, 5 ul of 10 x Universal Primer Mix (UPM), 1 Il of 10 M Gene Specific Primer 1 (GSP1) and 41.5 pl of Master Mix (34.5 pl PCR grade water, 5 pl 10X Advantage 2 PCR Buffer, 1 Il 10 mM dNTP Mix, and 1 pl 50X Advantage 2 Polymerase Mix). Positive and negative controls were prepared according to protocol. Gene-Specific Primer 1 (GSP1), 5'CTTCATCCTCCAATCGGTGT3' (SEQ ID NO:21), and Gene-Specific Primer 2 (GSP2), 5'GGTCGCTCAGAAGACGGTAC3' (SEQ ID NO:22), were designed from Btl 1-clone 33 with a Tm of 60 0 C and 64°C, respectively. Amplification was performed by 25 cycles using the PTC-100 [Trademark] Programmable Thermal Controller (MJ Research Inc, USA). Each cycle consists of 94 0 C for 5 seconds, 65 0 C for 10 seconds and 72 0 C for 3 minutes. 5' RACE products were initially analyzed by agarose gel electrophoresis using 1% w/vagarose/EtBr gel. Positive RACE products were amplified using the same parameters mentioned above and was resolved using agarose gel electrophoresis followed by purification using the GenElute Spin Columns (Supelco 56500, USA). DNA samples were then precipitated by phenol/chloroform extraction.

Aeceived 12 December 2001 -59- Purified 5' RACE products were finally analyzed by DNA sequencing using the ABI 377 DNA Sequencer (Applied Biosystems).

EXAMPLE 8 Hybridization of Btll-clone 33 and 5 '-RA CE product The full-length Btl 1 cDNA gene was generated by hybridizing a portion of the Btl 1-clone 33 with the 5'-RACE product. The portion of Btll-clone 33 used was an amplified segment of Btl 1-clone 33 sequence by PCR using GSP1 primer and T7 primer in a reaction consisting of 1 l of clone 33 plasmid DNA (100 ng), 1 Il of GSP1 primer, 1 tl T7 primer, 0.5 ll of 10mM dNTPs, 2.5 pl of 10X buffer, 0.375 ll of enzyme (Expand High Fidelity PCR System; Boehringer Mannheim, USA) and 18.625 ul of sterile water.

Amplification was performed using the temperature profile: 94°C for 2 minutes followed by 9 cycles of denaturation at 94 0 C for 15 seconds, annealing at 55°C for 30 seconds and extension at 72 0 C for 1 minute with a touchdown profile of 19 cycles at 94 0 C for seconds, 55°C for 30 seconds and 72 0 C for 1 minute with an increase of 20 seconds per cycle prior to a final extension at 72 0 C for 7 minutes. The amplified sample was purified as described for RACE product Hybridization of the 2 strands was performed by PCR using 100 ng each of the purified Btll-clone 33 segment cDNA and 5'-RACE product DNA. A PCR reaction mixture of 25 ul (composition as mentioned in F) was prepared using the Nested Universal Primer (NUP), 5'AAGCAGTGGT-AACAACGCAGAGT3' (SEQ ID NO:23), and the T7 primer, 3'CGGGATATCACTCAGC-ATAATG5' (SEQ ID NO:24). The DNA strands were denatured at 94 0 C for 30 seconds followed by annealing at 72 0 C for 5 minutes. Amplification was performed by 30 cycles using the PTC-100 [Trademark] Programmable Thermal Controller (MJ Research Inc., USA). Each cycle consists of 94 0 C for 30 seconds, 55°C for 30 seconds and 72 0 C for 3 minutes. PCR products were initially analyzed by agarose gel electrophoresis using 1% w/v agarose/EtBr gel. The PCR products were purified as described earlier and was finally analyzed by DNA sequencing using ABI 377 DNA Sequencer (Applied Biosystems).

Received 12 December 2001 EXAMPLE 9 Cloning of Full-Length Btll cDNA Gene The full-length Btl 1 cDNA gene was ligated into pCR2.1-TOPO cloning vector and was subsequently transformed into TOPIO One Shot [Trademark] competent cells using the TOPO TM TA Cloning version E (Invitrogen). In order to do this, a single 3'-A overhang was introduce to the full length Btl 1 by incubating in a 100 ul reaction consisting of 1 lg of purified DNA (20 ul), 2 ul 10 mM dATP, 10 pl of 10 x PCR buffer, 6 ul 25 mM MgC1 2 1 pl of (5 u/l) Taq Polymerase (Promega, USA) and 61 ul of sterile water at 72 0 C for minutes.

The reaction was then purified using the GFX [Trademark] PCR DNA and Gel Band Purification Kit (Pharmacia). The 100 pl reaction mix was added to 500 ul of Capture Buffer. It was mixed thoroughly and transferred into one GFX column inserted into a collection tube. This was centrifuged at 14,000 g for 30 seconds. The flow through was discarded and the column was washed with 500 pl of Wash Buffer by repeated centrifugation. The column was then transferred into a clean 1.5 ml microcentrifuge tube and the DNA was eluted in 50 pl of elution buffer by centrifugation at 14,000 g for 1 minute after incubation at room temperature for 1 minute. Ligation was carried out with 2 ul of the purified full length Btl 1 with 3'-A overhang added to 2 ul ofdd water and 1 tl of pCR2.1-TOPO vector. The ligation reaction was incubated for 5 minutes at room temperature. Transformation was then performed by mixing 2 Al of the TOPO [Trademark] Cloning reaction into a vial of TOP 10 One Shot [Trademark] competent cells which was previously treated with 2 pl of 0.5 M 1-mercaptoethanol. After incubating the One Shot [Trademark] transformation reaction for 30 minutes on ice, the cells were heatshocked at 42 0 C for 30 seconds in a water bath followed by 2-minute incubation on ice.

250 pl of SOC medium was then added to the reaction mixture prior to incubation at 37°C for 30 minutes in a horizontal shaker. Cells were then plated onto selective LB-ampicillin plates and was incubated overnight at 37 0 C. Transformed colonies, white colonies, were subcultured overnight at 37 0 C on LB medium with ampicillin. Plasmid DNA purified by c C

I

Received 12 December 2001 PX)PWEAPMnL3 d 12/12 -61- Wizard Plus SV minipreps DNA purification system (Promega) were analyzed by DNA sequencing using the ABI 377 DNA Sequencer (Applied Biosystems).

EXAMPLE Transformation of Full-Length Btll cDNA Gene Cloned full-length Btl 1 cDNA gene was purified by plasmid purification using the Wizard Plus SV minipreps DNA purification system (Promega). The full-length Btl 1 cDNA gene was amplified by PCR using designed primers to create restriction enzyme sites for expression vector plasmid. Primer Bt11CS2f, 5'CGGGATCCATGGCGGCTCGATCAGCA3' (SEQ ID NO:25), was used to create a BamH1 restriction site on the 5'end while Primer BtllR2, (SEQ ID NO:26), was used to create an Xhol restriction site at the 3' end. Pfu polymerase was used in the amplification process. PCR was performed for 30 cycles consisting of 94 0 C for 15 seconds, 55°C for seconds and 72 0 C for 1 minute using the PTC-100 [Trademark] Programmable Thermal Controller (MJ Research Inc). Gel-purified PCR product was digested with BamH1 and Xho 1. Restriction enzyme digested insert was purified by agarose gel electrophoresis followed by purification using GenElute spin columns (Supelco 56500) and phenol/chloroform extraction. Purified Btll insert was ligated overnight at 16 0 C into PGEX-4T-1 vector (Pharmacia Biotech) previously digested with BamH1 and Xhol.

Ligated Btl insert and PGEX-4T-1 vector was transformed into E. coli BL21 strain by heat-shock method. A reaction mixture of 200 pl of thawed competent E. coli BL21 strain with 10 ng of ligation mixture was incubated on ice for 30 minutes. The reaction was heatshocked at 42 0 C for 2 minutes and 30 seconds in a water bath followed by incubation on ice for 10 minutes. 800 ul of LB broth was then added to the reaction mixture which was incubated for one hour at 37 0 C. Cells were plated onto selective LB-ampicillin plates and was incubated overnight at 37 0 C. Transformed clones were subcultured in LB broth with ampicillin. Glycerol stock of the transformed clones were stored at -80 0 C for future use.

Received 12 December 2001 -62- EXAMPLE 11 Protein Expression and Purification ofBtll The expression of recombinant Btll clone as a fusion protein with glutathione Stransferase (GST) was performed by isopropyl-/3-D-thiogalactopyranoside (IPTG) induction. A 10X dilution of overnight culture of transformed clones was incubated approximately for 1 hour at 37 0 C in a shaker until the OD at 600 nm reaches 0.6. The expression was induced with 100 mM IPTG for 3 hours at 30 0 C. 300 il of the culture was taken before and after IPTG induction. Collected samples were pelleted and cells were resuspended in 25 gl sample buffer. Denatured samples were analyzed by SDS-PAGE.

Purification of the expressed protein was performed by affinity chromatography using the Glutathione Sepharose Column (Sigma). IPTG-induced bacterial pellet resuspended in trisbuffered saline (TBS) was sonicated using the Ultrasonic Processor XL (Heat Systems).

Upon centrifugation at 17,000 g for 30 minutes, the supernatant was collected and loaded onto the Glutathione Sepharose Column and was eluted with 10 mM of Glutgathione elution buffer as suggested by the GST Purification Module (Pharmacia Biotech). Purified protein samples were analyzed by SDS-PAGE and quantified by DC Protein Assay (BioRad).

EXAMPLE 12 Dot blot immunoassay The allergenecity of the recombinant Btll was determined in vitro by dot blot immunoassay using sera from mite sensitive patients. In brief, nitrocellulose membrane (Hybond C, Amersham Life Sciences) was cut to size, soaked in IX PBS (pH 7.4) and place on top of 3 mm Whatmann paper before finally being clamped into a BioDot apparatus (Bio-Rad). Two gg of purified Btl suspended in 200 gl PBS was applied per dot and drawn through the nitrocellulose by suction with a vacuum pump. Five gg per dot of crude Bt protein extract was used as positive control while 2 gg of purified GST was used as negative control. All dots were done in duplicates. The nitrocellulose was then removed and blocked with 5% w/v non-fat milk powder in PBST (PBS with 0.05% v/v Aeceived 12 December 2001 IN-= I lrrr~lll~ -63- Tween 20) for 1 hour at room temperature, followed by 6 washes with PBST for 5 minutes Pech. The nitrocellulose was then cut into various strips and labelled accordingly. The strips were incubated overnight at 4 0 C with 5X dilution of human sera in PBST. After thorough washing with PBST (6X for 5 minutes each), the membranes were incubated with biotinylated anti-human IgE (2 utg/ml) for 1 hour at room temperature followed by 6 washes with PBST for 5 minutes each. The membranes were then incubated with 5000X dilution of ExtrAvidin (peroxidase and alkaline phosphatase conjugated) for 1 hour followed by 6X wash with PBST. Positive reactions were detected using the ECL+PLUS (Amersham Pharmacia Biotech). Membranes were exposed to negative BioMax film (Kodak) and was developed according to the specifications of the manufacturer. These membranes were briefly washed with PBST and finally incubated in a developing solution containing BCIP and NBT in 10 ml of 1X AP buffer.

EXAMPLE 13 Identification and characterization ofBtll Screening of approximately 6 x 104 plaque forming units (pfu's) from the Bt cDNA library resulted to 36 positive plaques using a combination of DIG-labeled Df 11 fragment 2 and fragment 4 as molecular probes. Four of these plaques, designated as clone 2, clone 8, clone 32 and clone 33 were purified to homogeneity by secondary and tertiary screening process. Nucleotide sequence analysis revealed that the four clones are identical with different degree of truncations on the 5' end. The nucleotide sequence of the 4 clones showed polyadenylation sequences. CLUSTAL W Multiple Alignment Program revealed that the 4 clones exhibited significant alignment to Df 11 nucleotide sequence.

Out of the four clones, clone 33 exhibited the longest size comprising of 2,598 bp with a polyadenylation sequence from position 2581 to 2598. Alignment of the nucleotide sequence of clone 33 with Df 11 nucleotide sequence revealed 89% sequence homology as shown in Figure 2. Clone 33 nucleotide sequence revealed the absence of any methionine sequence indicating therefore that it is truncated on the 5'end. Several batches of screening process failed to isolate the full-length Btl gene by colony/plaque hybridization. The Aeceived 12 December 2001 .Op* EX.pca%237O&bLl l d 112101 -64- Rapid Amplification of cDNA Ends (5'-RACE) was used to fish out the full-length clone using designed gene specific primers (GSP's) based on the nucleotide sequence of the truncated clone 33.

Figure 3 shows the nucleotide sequence and the deduced amino acid sequence of the fulllength Btl cDNA gene after the hybridization of clone 33 and 5'-RACE product. The full-length Btl 1 cDNA gene is composed of 3111 nucleotide base pairs with a 2625 base pairs open reading frame from position 326-2950. An ATG (Met) sequence is found at position 326-328 while a TAA (stop) codon is found at position 2951-2953. The fulllength sequence shows a 325 nucleotide base pairs of untranslated region on the 5' end and a 158 nucleotide base pairs of untranslated region on the 3'end. The gene codes for an 875amino acid protein with a methionine on the N-terminus.

Figure 4 shows the amino acid sequence alignment of Btl with several paramyosins isolated from different invertebrates. CLUSTAL W Multiple Sequence Alignment program shows a significant homology ofBtl 1 to Df 11 and 3 other invertebrate paramyosins. Btl 1 amino acid sequence shows 90% sequence homology with the Dermatophagoides farinae paramyosin (Df 11). Moreover, the Btl 1 amino acid sequence exhibited 58% homology to the Drosophila melanogaster paramyosin, 50% homology with Onchocerca volvulus paramyosin and 49% homology to Caenorhabditis elegans paramyosin. Results of Basic BLAST search using the BLASTP 2.0 program with the National Center for Biotechnology Information (NCBI) databank likewise revealed a significant homology of between Btl gene and other invertebrates paramyosins such as Dirofilaria immitis, Brugia malayi, Schistosoma mansoni, Taenia soleum and Schistosomajaponicum.

Results of the human IgE plaque immunoassay using clone 2 (truncated clone) showed that out of 23 sera tested, 9 showed positive IgE reactivity. Table 4 summarizes the results of the IgE plaque immunoassay.

i Received 12 December 2001 I pa .pVPUWWWt bL=N370. amadM.~i2 TABLE 4 Summary of the results of human IgE plaque immunoassay Positive Negative Total Sera Tested Singapore sera 2 10 12 Taiwan sera 7 4 11 Dot blot immunoassay using the full-length Btl showed 68% positive reaction with human IgE from the Singapore sera tested (13/19) while only 30% positive reactions were observed from the Taiwan sera screened. All sera used are positive for IgE reactivity with the crude mite extract. Table 5 summarizes the result of the dot blot immunoassay. Figure 6 shows the representatives of the dot blot immunoassay.

TABLE 5 Summary of the results of dot blot immunoassay Positive Negative Total Sera Tested Singapore sera 13 6 19 Taiwan sera 11 26 37 A polymorphic variant of the Btl gene were isolated by Rapid Amplification of cDNA Ends (RACE) using mRNA extracted from mite cultures. The nucleotide and deduced amino acid sequences of the variant designated as Clone 1.2 is summarized in Figure 7.

"Examples 14 to 27 relate to Btl EXAMPLE 14 Immunization of mice Each mouse was immunized with 25 tug recombinant Dp 10 with the present of CFA (Complete Freund's Adjuvant). Mice were further challenged with Dp 10 and IFA Received 12 December 2001 -66- (Incomplete Freund's Adjuvant) at day 14 and 21. ELISA was performed to determine the titer of IgG produced.

EXAMPLE cDNA library screening Screening of the B. tropicalis Uni-ZAP cDNA library was performed by plaque immunoassay methods, using mouse anti-Dp 10 polyclonal antibodies prepared as described in Example 14. Phages were plated at 10,000 plaque-forming units (pfu) per 135mm petri dish. Plaques were lifted onto IPTG (isopropyl-3-D-thiogalactopyranoside) saturated nitrocellulose filters (Amersham). The filters were blocked in PBS containing 0.05% v/v Tween 20 (PBST) and 5% w/v non-fat milk. After washing, the filters were incubated with preabsorbed mouse serum diluted 1:500 in blocking solution overnight at 4°C with continuous shaking. The filters were then vigorously washed thrice with PBST, followed by incubation with alkaline phosphatase conjugated anti-mouse IgG (Sigma) at room temperature for an hour. The filters were then washed as before and were subsequently developed with BCIP/NBT (5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium). The positive plaques were picked and further screened for a purified clone.

EXAMPLE 16 DNA sequencing ofBtlO cDNA clone The BtlO-containing phagemid was excised in vivo using fl helper phage as described in the instruction manual of Zap-cDNA synthesis kit (Stratagene, La Jolla, CA). DNA was isolated and subjected to sequencing using ABI 377 DNA Sequencer (Applied Biosystems).

Received 12 December 2001 -67- EXAMPLE 17 Sequence analysis The sequences were aligned and homology analysis was performed using the BLAST network service provided by National Center for Biotechnology Information (NCBI).

EXAMPLE 18 Frequency of gE binding The frequency of IgE binding was determined by plaque immunoassay methods. A purified BtlO clone was plated out densely and were lifted onto IPTG saturated nitrocellulose filters (Amersham). Lifted filters were cut into sectors and probed with 13 mite-allergic individual sera, and 1 non-atopic serum as negative control. Bound IgE was detected with anti-human IgE alkaline phosphatase conjugated (Sigma).

EXAMPLE 19 Subcloning of BtlO inserts into expression vector The BtlO cDNA insert was released and ligated to pGEX-4T1 vector with BamHI and EcoRI restriction enzyme sites, and transformed to BL21. The fusion protein was produced by 1 mM IPTG induction and purified using glutathione Sepharose. BtlO was then subjected to cleaving by trombin. The IgE-immunoblotting was performed to determine the allergenicity of the fusion protein.

EXAMPLE SDS-PA GE and IgE immunoblot assay Immunoblots were prepared by loading about 200 ng/well of GST-BtlO and GST on a w/v SDS-PAGE, followed by electro-blotting onto nitrocellulose membranes (Hybond-C extra, Amersham Life Science, England). The allergenicity of BtlO was determined using IgE immunoblotting. Briefly, the blots were blocked in PBS containing Received 12 December 2001 -68- 0.05% v/v Tween 20 (PBST) and 5% w/v non-fat milk. After washing, each blot was incbhated with 600 ul of individual allergic serum diluted 1:5 in blocking solution overnight at 4°C with continuous shaking. The blots were then vigorously washed thrice with PBST, followed by incubating with 2 gg/ml of biotin-anti human IgE (PharMingen, San Diego, CA) at room temperature for an hour. The blots were then washed as before and were subsequently incubated with ExtrAvidin conjugated with peroxidase (Sigma) (1:4000) for another hour at room temperature. After washing, the signals were developed with ECL-PLUS western blotting detection system reagent kit (Amersham Life Science, England) and autoradiography.

EXAMPLE 21 Skin prick tests Skin prick tests were conducted according to the protocol previously described Briefly, the skin of the forearm's volar was pricked with a disposable lancet with the presence of an allergen droplet. The prick was regarded positive when a wheal diameter is greater than 3X 3 mm after subtracting the negative control 30 minutes after the prick. Glycerol-buffer and 1 mg/ml of histamine were used as negative and positive controls, respectively. 25 tg/ml of purified allergens were used in this study.

EXAMPLE 22 DNA sequence analysis Two positive plaques were obtained after screening of approximately 6 x 104 pfu from the Bt cDNA library, designating clone 1 and 2. These clones were further purified to homogeneity and the inserts were sequenced. Nucleotide sequence analysis revealed that clone 2 is truncated at the 5' end. The complete nucleotide sequence was obtained in the other clone, consisting of 1014 base pair (Figure including 6 bp long 5' non-translating region. The sequence has an ATG start codon at position 7 to 9, and a stop codon, TAA at position 859 to 861, which showed an open reading frame coding for a 284 amino acid molecule. The full length clone is designated "BtO" The corresponding protein is referred Received 12 December 2001 naop paans ror t2anM -69to as "Btl0". The nucleotide and corresponding amino acid sequence of BtlO are defined in SEQ ID NO:28 and SEQ ID NO:29, respectively.

EXAMPLE 23 Protein sequence analysis Protein sequence homology search was performed by using the Advance BLAST search from NCBI. The search results shows a very significant homology of BtlO to other invertebrates tropomyosin, especially from Lepidoglyphus destructor, Dermatophagoides farinae and Dermatophagoides pteronyssinus. Btl0 showed 96% identity to published Der f 10 and Lep d 10. Sequence alignment also showed BtlO shared 95% of identity to both of Der p 10 sequence. Figure 8 shows the alignments of the deduced amino acid of tropomyosin from various species of mites mentioned before. The alignments were performed using the CLUSTAL W Multiple Sequence Alignment package.

EXAMPLE 24 IgE plaque immunoassay Nine out of 13 mite-allergic individuals' sera used in this assay showed a positive result, which contributed to 69% of IgE reactivity. There is no IgE binding on the sector that incubated with non-atopic serum (Figure EXAMPLE Expression and purification ofGST-BtlO Expression of recombinant fusion BtlO clone gave a single band at around 60 kDa on a w/v SDS-PAGE (Figure 11). Lane 3 of Figure 11 is Btl0 that was ran at around kDa and being released by trombin digestion followed by glutathione agarose purification.

The calculated molecular weight of BtlO is 33.2 kDa, which is similar to Mag 44, as reported by Aki et al. (29).

J Received 12 December 2001 P-'vaE.*PatM13 71 n««l Ol*T*)H -I7ImI EXAMPLE 26 IgE-binding study of recombinant fusion BtlO The ability of IgE-binding of recombinant fusion BtlO was determined by Western Blotting (Figure 12). The results showed that the BtlO fusion produced is able to bind IgE of mite-sensitized individual significantly (Lane 4, Figure 12). Individual B also reacted to degraded product of GST-BtlO, which ran at lower molecular weight, shown in lane 4, blot B, Figure 12. The IgE-binding band at around 35 kDa in Bt crude extract might be the corresponding protein that is present in nature (Lane 2, Figure 12). Non-fused GST was loaded next to fusion BtlO in order to show the IgE reactivity is specific to BtlO but not GST (Lane 3, Figure 12). The two high molecular weight proteins was due to the nonspecific binding as indicated by negative control (Lane 1, blot C, Figure 12).

EXAMPLE 27 Skin Prick Test The trombin-cleaved Btl0 was tested in vivo by the skin prick test. Thirteen out of 109 mite-sensitized volunteers showed positive reaction to Btl0, which contributed to approximately 12% of the volunteers tested. Fifty-four of the volunteers were also tested for shrimp reactivity and 11 of them reacted positively. Three of these shrimp-positive individuals also have positive SPT to Btl0, which might be due to the cross-reactive allergen, tropomyosin "Examples 28 to 39 relate to BtA2" EXAMPLE 28 Serum samples Sera from 4 adults show showed positive skin prick test reaction to B. tropicalis extract were pooled and used to screen the B. tropicalis cDNA Xgtl library by plaque IgE immunoassay.

Received 12 December 2001 -71 EXAMPLE 29 Screening of )gT1 exprssion library ofB. Tropicalis Library screening was performed by plaque IgE immunoassay. The pooled sera was adsorbed with lysate prepared from E. coli (Y1090) and recombinant E. coli containing overnight at 4 0 C on an orbital rotator before use. Phages were plated at 5-10,000 plaque-forming units (pfu) per 145 mm petri dish. Plaques were transferred to nitrocellulose filters (Amersham) that were saturated with isopropyl-g-Dthiogalactopyranoside (PITG). After blocking the filters with 5% w/v solution of non-fat milk powder for 1 hour, they were then washed three times with PBS containing 0.05% v/v (PBST), followed by overnight incubation with sera (1:1 dilution in PBST with 1% w/v non-fat milk and 0.02% w/v sodium azide) at 4 0 C. These filters were again washed three times. They were then incubated with the monoclonal anti-human IgE that was alkaline phosphatase conjugate (1:1,000 dilution) for an hour at room temperature followed by three washes in buffer. After the last wash, the filter is blotted on paper towels and then incubated in developing solution containing 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) and nitro blue tetrazolium (NBT, Sigma), for half to an hour. An intense bluish purple colour reaction signal indicates a positive reaction. The clone that reacts positively in the first round was subjected to several additional rounds of plating and screening to obtain a pure single plaque. The frequency of IgE reactivity of the clone was carried out using sera from the mite allergic patients. The frequency study was performed using the plaque IgE immunoassay as described above.

EXAMPLE Human IgE reactivity ofBtA2 clone ofB. tropicalis To study the frequency of IgE reactivity of clone BtA2 in the population, the following experiment was performed. 19 sera from candidates with positive skin prick test to B.

tropicalis extract and a negative control serum were taken. Individual serum from each candidate was adsorbed with lysate prepared from E. coli (Y1090) and recombinant E. coli Received 12 December 2001 ropstEpau ld37il.rK l l i217112 -72containing GST-B t5 (1:1 dilution) overnight at 4°C on an orbital rotator before use. BtA2 Xgt 11 clone was plated at approximately 1,000 plaque-forming units (pfu) per 145mm petri dish. Plaques were transferred to nitrocellulose filters (Amersham) that were saturated with isoprop7yl-1-D-thiogalactopyranoside (IPTG). After blocking the filters with 5% w/v solution of non-fat milk powder for 1 hour, they were then washed three times with PBS containing 0.05% v/v Tween-20 (PBST), followed by overnight incubation with sera (with 1% w/v non-fat milk and 0.02% w/v sodium azide) at 4 0 C. These filters were again washed three times. They were then incubated with the monoclonal anti-human IgE that was alkaline phosphatase conjugate (1:1,000 dilution) for an hour at room temperature followed by three washes in buffer. After the last wash the filter was blotted on paper towels and then incubated in developing solution containing 5-bromo-4-chloro-3-indolyl phosphate (BCIP, Sigma) and nitro blue tetrazolium (NBT, Sigma), for half to an hour. An intense bluish purple colour reaction signal indicates a positive reaction (Figure 17).

EXAMPLE 31 Dot blot immunoassay The pattern of reactivity of allergie sera against BtA2, GST-BtA2, GST-Bt5 and GST were analyzed by dot blot. A total of 5 sera were used. These sera were taken from candidates with positive skin prick test with B. tropicalis extract. Nitrocellulose was cut to size, soaked in PBS (pH 7.4) and placed on 3 mm Whatmann paper before finally being clamped into a BioDot apparatus (Bio-Rad Laboratories) Samples with 2 pg of BtA2, GST-BtA2, GST-Bt5 and GST, and 5 ug of B. tropicalis extract were applied in a volume of 200 gl and drawn through the nitrocellulose by suction with the help of a vacuum pump.

The nitrocellulose paper was then removed and blocked with 5% w/v non-fat milk powder in PBST (PBS with 0.05% v/v Tween 20) for 1 hour at room temperature, followed by three washes with PBST, 10 minutes each. The nitrocellulose paper was cut into various strips and labelled accordingly. These strips were incubated with their respective sera overnight at 4 0 C in a shaker. After which, these strips were washed three times, 15 minutes each with PBST at room temperature; incubated with biotin anti-human IgE (2 jig/ml) for 1 hour at room temperature followed by another 3 washes of 15 minutes each; incubated Received 12 December 2001 -73with ExtrAvidin peroxidase and ExtrAvidin alkaline phosphatase for further 1 hour also at room temperature and a final 3 washes with PBST of 15 minutes each. The ECL+PLUS (Amersham Life Sciences) western blotting detection system reagent kit was used.

Negative film (Kodak) was exposed to the nitrocellulose paper and developed according to the specifications of the manufacturer (Figure 19).

EXAMPLE 32 Isolation ofDNA from )gtll cDNA clone Phages from immuno-positive plaques were amplified by the liquid lysate method. DNA was isolated using the Magic Lambda Preps DNA Purification System kit (Promega) according to the manufacturer's instructions.

EXAMPLE 33 Polymerase chain reaction amplification Approximately 40-60 ng of DNA was added in a 25 tl reaction mixture containing 0.4 pmol of Xgtl21 forward and reverse primers, 0.2 mM of dNTPs, 1U of Native pfu DNA polymerase, 2.5 jul of 10X Native pfu DNA polymerase buffer (Stratagene). Forty cycles of amplification was performed using a GeneAmp PCR System 2400, Perkin Elmer. Each cycle consists of 95°C for 1 minute 30 seconds, 65 0 C for 1 minute 30 seconds and 72°C for 2 minutes. The PCR DNA was further purified after gel electrophoresis using QIAEII Gel Purification Kit (150), Qiagen.

EXAMPLE 34 DNA sequencing to 80ng of the amplified DNA was added to a 20 tl reaction mixture containing 0.16 pmol of either Xgtl 1 forward or reverse primer and 8 al of BigDye terminator RR mix (PE Applied Biosystems), following the detailed protocol of the manufacturer. 25 cycles of Received 12 December 2001 ^OpaiEq pda23f 7 bL3imlmlp l s2i1i2i01nH lK -74amplification were performed in a Gene Amp PCR System 2400. Each cycle consists 96 0

C

for 30 seconds, 50 0 C for 30 seconds and 60 0 C for 4 minutes.

EXAMPLE Expression of BtA2 recombinant protein The BtA2 was successfully cloned in the pGEX system for recombinant protein expression in E. coli (Pharmacia) as well as in Pichia vector (pPIC9) for expression as a secreted protein in Pichia pastoris (Invitrogene) following the protocols of the manufacturers.

EXAMPLE 36 Expression of recombinant BtA2 clone in E. coli The BtA2 clone was amplified by PCR using the primers CGGGATCCGCTCTCGACTTTACCAGC-3' (BamH1 site in italics) [SEQ ID NO: 76] and CGGAATTCTTAACCCCTGGAGGGCAG-3' (EcoR1 site in italics) [SEQ ID NO: 77], the PCR product was cut with BamH1 and EcoR1. The purified clone BtA2 from BamH1 and EcoR1 digest was ligated to pGEX-4T1 vector (Pharmacia Biotech) and transformed into E. coli BL21. Recombinant clones were confirmed by restriction analysis of a miniprep from transformed BL21. Bulk culture of recombinant was grown in Luria broth containing 100 lg/ml of ampicillin to log phase and then induced with 1 mM of isopropyl /-D-thiogalactopyranoside (IPTG). Cells were harvested 2 hours later and washed once with TBS, pH 7.4. Pelleted cells were thoroughly sonicated in TBS, pH 7.4 in the presence of 1 mM phenylmethylsulfonyl fluoride (PMSF), 20 gg/ml of DnaseI and 1% v/v Tween 20, and centrifuged. The supernatant was applied to glutathione sepharose (Sigma) column followed by elution with 10 mM reduced glutathione in 50 mM Tris, pH as suggested by the GST purification Module (Pharmacia Biotech). The GST-BtA2 protein was cleaved with the enzyme thrombin (1 Unit thrombin per 40.D. of the fusion protein at 280 nm wavelength) in the presence of 1 mM of CaC12 with gently shaking at 4°C overnight. The reaction mixture was then passed through the glutathione sepharose column and the elute was the 7 kD recombinant protein BtA2 (Figure 18, Gel GST was Received 12 December 2001 bound to the column and it could be eluted out by the addition of 2 ml of Glutatione Elution Buffer (10 mM reduced glutathione in 50 mM Tris, adjust the pH to 8.0) per ml of column matrix.

EXAMPLE 37 Expression of recombinant BtA2 clone in the yeast Pichia Pastoris The BtA2 clone was amplified by PCR using the primers GAAAAGAGCTCTCGACTTTACCAGC-3' [SEQ ID NO: 78] and CGGAATTCTTAACCCCTGGAGGGCAG-3' (EcoRl site in italics) [SEQ ID NO: 79], then the PCR product was cut with EcoR1. It was subjected to electrophoresis and further purified using QIAEII Gel Purification Kit (150), Qiagen. It was ligated into P. pastoris expression vector pPIC9 (Invitrogen) for extracellular expression. The vector pPIC9 was previously digested with Xhol and then treated with Klenow enzymes, and finally digested with EcoR1. After ligation, it was transformed into P. pastoris. GS115 (Invitrogen), screening for recombinant BtA2 protein producing clones according to the protocols of the manufacturer. The Pichia host strain GS115 has a defect in the histidinol dehydrogenase gene (His4) which prevents it from synthesizing histidine. All expression plasmids carry the wild type His4 gene which complement His4 in the host, so transformants are selected for their ability to grow on histidine-deficient medium. Transformants ofGS1154 with Bgl II-linearized pPIC9 construct favour recombination at the AOX1 locus. Displacement of the alcohol oxidase (AOX1) structural gene occurs at a frequency of 5-35% of the His+ transformants. By patching or replica-plating on minimal dextrose (MD) versus Minimal Methanol (MM) plates, Mut+ and Mut- (methanol utilization slow) transformants can be readily distinguished. Several transformants were then grown overnight in 1 ml of BMGY media w/v yeast extract, 2% w/v peptone, 100 mM potassium phosphate, pH 1.34% w/v yeast nitrogen base with ammonium sulfate, 0.00004% w/v biotin, 1% v/v glycerol). Overnight cultures were then transferred to a larger volume and grew to O.D. 2- 6 at 600 nm wavelength to los phase. Then the cells were spun down and resuspended in BMMY medium v/v methanol replaces the glycerol of BMGY medium) with its volume reduced to 1/10 of its earlier volume in BMGY. The culture was allowed to grow Received 12 December 2001 -76for several days. 1 ml of supernatant was taken daily from the culture to screen for recombinant protein expression and at the same time, 100% v/v methanol was added to a final concentration of 0.5% to the culture. The recombinant protein in the supernatant was analysed in a SDS-PAGE (10% w/v) (Figure 18, Gel A suitable clone was selected to grow in large volume for mass extraction of recombinant A2 clone protein.

EXAMPLE 38

SDS-PAGE

Recombinant proteins were separated by normal tricine SDS-PAGE. The cathode buffer was 0.1 M Tris, 0.1 M Tricine, 0.1% w/v SDS, pH 8.25 (adjust pH with Tris or Tricine) and the anode buffer was 0.2 M Tris, pH 8.9 (adjust the pH with 6N HCL). Samples were denatured and reduced by boiling the samples in a 0.1% v/v mercaptoethanol, 4% w/v SDS, 0.01% w/v Serva glue G, 12% v/v glycerol and 50 mM Tris, pH 6.8. The samples and the molecular weight marker (14-97 kD, BioRad) were applied to the gel and the electrophoresis was performed at 20 mA/gel (approximate voltage 100 Volts) until the bromophenol blue reached the bottom of the gel. Gels were stained with Coomassie brilliant blue R-250 (Sigma).

EXAMPLE 39 Sequence analysis The deduced nucleotide sequence was submitted to the databases of the National Center for Biotechnology Information (NCBI), using the BLAST network server for sequence homology search. The amino acids sequence homology search was carried out simultaneously.

The nucleotide sequence of the clone is 507 bp in length. This includes a linker sequence ggccagag, a 286 bp 3' untranslated region with a poly-A tail, and a 218 bp probable truncated coding region for the recombination protein with a stop codon (TAA) at nucleotide residues 219-221. The deduced peptide has a cycteine rich between nucleotide Received 12 December 2001 -77residues 70 to 200 as detected in the databases of the National Center for Biotechnology Information (NCBI), using the BLAST network server. The inferred amino acid sequence from nucleotides 9-218 indicated that this partial clone codes for a protein of 66 residues, with 8 cysteine residues in the molecule (Figure 16).

"Examples 40 to 47 relate to Bt5 polymorphic variants" EXAMPLE PCR amplification and cloning of Bt5 genomic gene Genomic DNA sequence for Bt5 was cloned by direct PCR amplification of storage mite, Bt obtained from Singapore and Columbia. Briefly, numerous of mites were suspended in 1 X PCR reaction buffer (10 mM KC1,10 mM (NH4) 2

SO

4 20 mM Tris-Cl (pH 8.75), 2 mM MgSO 4 0.1% Triton X-100, 100 gg/ml BSA) and boiled at 100 0 C for 10 minutes.

The sample was cooled on ice for 10 minutes. The PCR reaction was carried out in 1 X PCR buffer containing 100 mM of dNTP, 20 pmoles of each sense and antisense primers and 2.5 units ofpfu in a final volume of 100 tl. The sense primer and the antisense primer are 5'CCCGGATCCACAATGAAGTTCGCCATCGTTCTT3' SEQ ID NO:35 (primer) and 5'GCTCTAGATTATTGGTTTGAATATC3' SEQ ID NO:36 (primer), respectively.

The reaction was heated to 95°C for 5 minutes, annealed at 57 0 C for 2 minutes, and polymerized at 72° for 2 minutes followed by 40 cycles of amplification, each consisting of 95°C for 1 minute, 57°C for 1 minute, and 72 0 C for 2 minutes. The final step of the last cycle extended 72 0 C for 10 minutes. One hundred fold dilution of the pfu amplified sample was reamplified using Taq polymearse. The gel purified PCR fragment was then cloned into the pCR2.1 vector using the TA cloning kit (Invitrogen). Sequence of the Bt5 genomic gene was determined by ABI PRISMTM DNA sequencing kit.

I

Received 12 December 2001 P:XapcjM\z mk« 12nlli2MinI *IIIM -78- EXAMPLE 41 Preparation of antigens Purification of recombinant The recombinant allergens Bt5 has been purified using recombinant E coli as a fusion with glutathione -S-transferase (GST). The proteins were purified by affinity chromatography using glutathione agarose beads. The purified proteins were then cleaved by thrombin digestion. The cleaved proteins were separated from the GST fusion partner by another round of affinity chromatography using the glutathione agarose beads and then analysed on SDS-PAGE gel. Proteins were filter-sterilized before use.

Preparation of crude mite extracts Lyophilized Bt mites were homogenized in the presence of liquid nitrogen. The homogenised mite proteins were extracted in PBS buffer for 48 hours at 4 0 C. The extracts were analyzed on SDS-PAGE gel and then filter sterilized before use.

EXAMPLE 42 Establishment of T cell lines from allergic individuals Ten allergic individuals have been selected on the basis of their skin test reactivity. These individuals were skin test positive for Bt. The PBMC from these individuals have been collected and the T-cells cultures were first set up using Bt crude extracts, the second round of antigen stimulation was performed with recombinant Bt5 allergens. The antigenspecific T-cell lines have been established. Two cell lines have been used for the epitope mapping using a panel of synthetic peptides.

Received 12 December 2001 P LapU)kam3L nmnhfrka@m TOT WA-124IMI -79- EXAMPLE 43 T-cell epitope mapping Sets of peptides covering the entire sequences of Bt5 were made by the Multipin method by Chiron Mimotopes Pty., Clayton, Victoria, Auatralia. The purity of the peptides was confirmed by high performance liquid chromatography. The peptides were designed to be 16-amino acid residues in length, overlapping by 10 or 13 amino acid residues Peptides 67 are Bt5 peptides designed on the basis of published sequence of Bt5. Peptides 80-96 are the peptide variants of Bt5 (refer to Figure 20). These peptide variants contain naturally occurring mutations of Bt5 proteins. Subsets of adjacent peptides were pooled for initial screening, and only those peptide pools that were stimulatory were examined in further detail for the identification of particular epitopes. T-cell lines specific for Bt5, were generated from PBMC of ten allergic individuals. These cell lines were tested against the synthetic peptides by T-cell proliferation that was measured by thymidine uptake assay.

EXAMPLE 44 Cell proliferation assay T-cells (1 x 105) were cultured with or without irradiated antigen presenting cells (5 x 105) in a final volume of 0.2 ml in complete RPMI in the presence of varying concentration of protein antigens. Tridium thymidine ([3H]TdR) were added at 48 hours, 72 hours or 96 hours for 18 hours. Cultures were harvested and thymidine incorporation was analyzed by liquid scintillation.

EXAMPLE Analysis of Bt5 genomic sequences The inventors have shown that a small degree of sequence polymorphism of mite allergens exerts a significant impact on the host immune system, including both the humoral and cellular immune responses. As Bt5 is the main indoor allergen causing asthma in tropical and subtropical countries, it will be important to examine whether there is any residue r. IV uVIuuV i1.L I Received 12 December 2001 polymorphism exists in Bt5 gene(s). In accordance with the present invention, the inventors have cloned the genomic sequences of Bt5 gene(s). By aligning the coding and intron sequences, a total number of 10 Bt5 gene variants have been identified (Figure 21).

Sequences 1 to 5 and 6 tolO representing Bt5 gene sequences obtained from Colombian and Singapore Bt mites, respectively. Like Der p 2, there is only one intron present in However, the intron of Bt5 is shorter and its location is further downstream of the first codon, ATG, than Der p 2 gene The size of the intron is ranging between 54 to 56 bp with stretches of nucleotides composing of either A or T base. Among the 11 variants, nucleotide polymorphism are mainly found within the first half of the coding sequence (Table Some of the nucleotide changes resulted in some amino acid residue changes (Table The residue changes can be found as the conversion of a negatively charged residue to a neutral residue (residue a positive charge amino acid to a negative charge residue (residue a neutral residue to a negative charge residue (residue 12), a neutral residue to a neutral residue (residues 17, 23 and 33), or a neutral residue to a positive residue (residues 34 and 11). Other types of property alteration including hydrophobic residue to hydrophobic residue (residue 34), acid residue to aliphatic residue (residue basic residue to acidic residue (residue 12), aliphatic residue to aromatic residue (residue 17), amide residue to aliphatic residue (residue 23), aliphatic residue to basic residue (residues 34 and 117).

EXAMPLE 46 Sequence analysis of Bt5 cDNA clones The first cDNA sequence for Bt5 was published by Arruda et al. subsequently a partial sequence of Bt5 was reported by Carabolla et al. The inventors independently isolated Bt5 cDNA clones from a cDNA library constructed using total RNA extracted from local Bt mites. As shown in Figures 12 and 22, the instant Bt5 cDNA sequences differ from the published sequences in two important aspects. First, the cDNA clones revealed a number of polymorphic residues that were not previously reported. Secondly, the cDNA clones have extended and untranslated regions and there was a high Received 12 December 2001 -81degree of sequence diversity shown by the various cDNA variants in these untranslated regions (Figure 13).

EXAMPLE 47 T-cell epitope mapping In order to evaluate the immunological impact of the sequence polymorphisms found in the various Bt5 variants, T-cell epitope mapping was performed using a panel of Bt5 synthetic peptides (Figure 20). As shown in Figure 23, the T-cell epitopes recognized by allergic subjects are largely located in regions where polymorphic residues are found. A substitution of one or two amino acid residues resulted in significant difference in T-cell response. The results suggest that these polymorphic residues are potentially important for the design and development of effective immunotherapeutic reagents for the treatment of mite allergy. It has been reported that there was a small but significant degree of sequence polymorphisms exists in Der p 1 and Der p 2, the two major allergens for Dermatophagoides pteronyssinus mites (32,33). Further, the polymorphic residues of Der p 2 were located in regions containing major T-epitopes(32,33). Taking together, it is clear that the sequence polymorphisms exist in these major mite allergens have significant impact on the immune responses. Therefore, it is conceivable that these polymorphic residues are potentially important for the design and development of effective immunotherapeutic reagents for the treatment of mite allergy.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Received 12 December 2001 P OpTLpas337bL j t 2L~ 82

BIBLIOGRAPHY

I. Sporik RB, et.al., 1992, Clin Exp Allergy 22: 897-906.

2. indoor allergens and asthma. Report of the 3 rd International Workshop, Cuenca, Spain, June 30-July 2, 1995.

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4. Zhang et. al., 1997, Clin Exp Allergy 27:~ 876-885.

Chew et at., 1999, C/in Exp Allergy 29. 201-206.

6. Gabriel et. al., 1982, Clin Allergy 12: 157-71.

7. Arruda et. at., 199 1, Clin Exp Allergy 21: 43 3-9.

8. Puerta et. at., 1991, JAl/ergy C/in Immunot 88: 943-50.

9. Caraballo et. at., 1993, C/in Exp Allergy 24: 1056-60.

Stanaland et. at., 1994, 1 Allergy C/in Immunol 94: 452-7.

11. Fernandez-Caldas (1997) JIn vest Al/ergot C/in Immunot 7:402-4.

12. Fernandez-Caldas and Puerta, Sensitization to various mite species. In: Progress in Allergy and Clinical fimunology. Vol 3, Stockholm. 1995, 323-9.

13. Tee (1994) C/in Exp Allergy 24:63 6-40.

14. Arlian et. at., 1993, 1 Allergy C/in Immunol 91: 1042-1050.

Received 12 December 2001 83 Arruda et al., 1995, Int Arch Aleergy Immunol 107: 456-7.

16. Caraballo et. al., 1996, JAllergy C/in Immunol 98(3): 573-9.

17. Ari-uda et. al., 1997, American Journal of Respiratory and Critical Care Medicine 155:343-50.

18. Caraballo et.al., 1997, mnt Arch Allergy Immunol 112: 341-347.

19. Puerta et. al., 1996, JAllergy C/in Immunol 98(5): 932-937.

Thomas et. al., 1999, C/in Exp Allergy 29: 21. Tsai et. al., 1998, JAllergy C/in Immunol 102(2): 295-303.

22. Epton et al., 1999, mnt Arch Allergy Immunol 496: 1-7.

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24. Altschul et al., 1997, Nuc!. Acids Res. 25:3389.

Ausubel et al., "Current Protocols in Molecular Biology" John Wiley Sons Inc, 1994-1998, Chapter 26. Marmur and Doty, 1962, J Mo!. Biol. 5: 109.

27. Bonner and Laskey, 1974, Eur. J Biochem. 46: 83.

28. Jimenez, 1999, J Allergy C/in. Immunol. 103: S 185.

Received 12 December 2001 84 29. Aki et al., 1995,.!. Allergy Clin. Immunol. 96: 74-83.

Witteman et al., 1994, Int. Arch Allergy Immunol 105: 56-61.

31. Bronswijk et 1997, Acarologia 15: 477-89.

32. Chua et al., 1996, Clinical and Experimental Allergy 26: 829-837.

33. Chua et al., 1993, Int. Arch ofAllergy and Immunology 364-8.

34. Platt-Mills and DeWeck, 1989, J1 Allergy Clin. Immunol. 83: 4 16-72.

Chew et 1999, Clin. Exp. Allergy 29: 201-6.

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37. Shek et 1999,.!. Allergy Clin. Immunol. 103: 526.

EDITORIAL NOTE APPLICATION NUMBER 78918/00 The following Sequence Listing pages 1 to 86 are part of the description. The claims pages follow on pages 85 to 94.

WO 01/30817 WO 0130817PCT/AUOO/01227 -1I- SEQUENCE LISTING <110> National University of Singapore <120> Novel therapeutic molecules and uses therefore <130> 2337808/EJH <140> International <141> 2000-10-10 <150> 9905313-4 <151> 1999-10-26 <150> PQ8842 <151> 2000-07-18 <150> PQ8844 <151> 2000-07-18 <150> PQ8845 <151> 2000-07-18 <160> 79 <170> Patentln Ver. 2.1 <210> <211> <212> <213> 1 2598

DNA

Blomia tropicalis <400> 1 caaagaacaa ctcaggttga aatacaccgt ttaccgctca aatacaagat gtcggataag gagaagcaaa agttccaagc cgaagttttc gaattgttgg gactgccaac aaggacaagt tggtcgctca gaagacggta gaaaagttgg 120 acatgagttg aacattaaga ttgaagaaat caatcgaact gttgttgagg 180 ccgacaacga ttgtcgcaag agaattcgga attgatcaag gaagttcatg 240 ttcattggac aatgctaacc acttgaaggg acaaattgca caacaattgg 300 WO 01/30817 WO 0130817PCT/AUOO/01227 aggacacccg cc~c&;c&L i.

cccgattaga ccaaatacga tggcccagaa cattggaaaa aaaaggctac acctcgacct atctccgtgt accaaaagga aatctcaatt tggagaacga agaagaggca gaaacgattg aattgaacaa acgactcaag taataaggct gcttcaagcc tgtcactcaa ggaacaagca agtcaacgaa tgaattctct cgaacgagtg aacggaacgt tctccaaatc cgccaagttg acacgcagaa tcaaaacgag cgaaaaggt c cttgaccggt agctgaatcc agt tcctggc aacagcatgc aaacatagat ctcgattagc atgtaacaat aaataagaag aaaagcgtct acaccgattg at tggaa cga agccgaactt gatttcggaa gcaaaaggct cgcacatgcc caagagcaag caagattgca agcattggcc gaatgacgcc cgagaaaaat aagaatcaac gcacaaaagg ttgaacatgc aagaagtac aacatcgact cattatgatg cgacgatgCC cttcgtgcta cttacaacca gctcttcaaa caaaaactta gtcacaaaat cgaattgaag gaatcgcgaa actgaaaaga gaggaccata aaggtttaca gtccgacgat aatttgtcgt ggtactcgac aacaaattca ataaatctaa gtttaccacc caatgatttt agga tca aa a tgtataccag gaggatgaag tt-g-tnr caattgacta caagctcacg tatgaggaac cgactccaat caggcattgg ttggaggagg gacttgcaga cgtgagaaca caccgacgaa tgcttgccgc gattgactgc aagaggagat gattggctga aagca caaa t tgc agaagac aggtacatcg aagcattgac aacgagctgc tcaatgtcaa acgattacga ccattgaggt tggaaa caat aagttgaggc tccgtgatgt tgcttcgaaa aacaaattca accgccaaat tccaacaaga tcatccgagc aagtct ttgt ttatttcata taaaaaaagt actatcacca acaaaacaaa taagatgtca atatacattt aacgaaaacg tn, anitac aagctaatgg ttgatgaggt aattggaagc cggaggtaga agaaacgcgt tcagcatgtt aattgcaaca agaaactcgc tccatgaaca atccaaggaa cgagt tggca tgaagctctt ggctgaagct cactgaattg gatcaagaag tcaattgcaa cgccgaattg cgaacaaatg cctcgcttcg tgaagtacac caagtcgaca caagaaatca taatgcattg tgagattgag gaaggatcat attgttgcaa gcaagaacag gttggaagcc caaacatcga cacggaacaa taaatcaaac tttaaaaaaa cctccaaccc aaaatgccca gaagtataaa aaaataccaa atcaagcttg attggaaaa cgatgctgct tgaagaactt attgttgaac agttttgatc t tcccaattg gttggaacaa cgagtacgaa tgatgatttg agagatcgag cttgaaacct cagacacgac cgaaaacaat aaattgaaga gaattgtcgt caagctctcc caagctgttg gaggagatgc cacgaagagg gccaagagca aaggaact cc aaggacttgc ttggaaaccg gctggtggta gttgaagaag cgtgtcaagg gaaatgtccg gagggaatga gccgaagacc tcgtgggtca gagagctcga aataatttga tcacaacctc tggttatata aatgaattcc aacaacaata taaaattatt gagaaccacg gaatctiaci tcatggaaat cgacgaaaga aaatgtagtg atggacttgg gagaagatca actcaaaagg aagttgcgag gctgaagcta atcaaaccat tgcgaaaaca acgactatga accaaattga ccgaggttgc tggatgccgc aaattactga atcaattggg gagtcaactt cagttgtccg aattggagac gaatctctga tcgaatcaga aagtacgaaa aacgagtcat aacgacgacg aattgttgct ataaattgaa gccaacaaaa gagctgatca ccaccagtca acttttaaat aattcaaacc ttaaacacac tatagcatca atctaaaaac ccaaccaaag tattatcctt 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 WO 01/30817 WO 0130817PCT/AUOO/01227 aaaaaaaaaa aaaaaaaa 2598 <210> 2 <211> 3111 <212> DNA <213> Blomia tropicalis <220> <221> CDS <222> (2)..(3109) <400> 2 g aca tat tcg aat caa act ctc gtc tag tga gtt gtg cac tcg tcg tcg 49 Thr Tyr Ser Asn Gin Thr Leu Val Val Val His Ser Ser Ser agt att cga Ser Ile Arg ctc acc aca Leu Thr Thr aac aac aag gtg Asn Asn Lys Val caa tac ata Gin Tyr Ile cac aca tac aca His Thr Tyr Thr cac tgc att cgc His Cys Ile Arg cat att cga cct His Ile Arg Pro cca agt ggc gta Pro Ser Gly Val aag tct Lys Ser so ctc aca tca tct Leu Thr Ser Ser cca gaa gca gag Pro (flu Ala Glu tag tag acg atc aac Thr Ile Asn 193 atc agt ctc atc Ile Ser Leu Ile ctt cta aag cat Leu Leu Lys His tga aca cct ttc tac Thr Pro Phe Tyr aat tta caa gtg Asn Leu Gin Val acc att ttc caa Thr Ile Phe Gin taa gac ata agg gaa tag aac Asp Ile Arg Giu Asn caa tat tga ttt gat ttg aaa caa taa tca acg aca atg gcg gct cga WO 01/30817 WO 0130817PCT/AUDO/01227 Gin Tyr Phe 100 Asp Leu Lys Gin Ser Thr Thr Met Ala Ala Arg 110 tca gca aag Ser Ala Lys 115 tat atg tat cag tcc agt cgt get gga cac gge ggc gac Tyr Met Tyr Gin Ser Ser Arg Ala Gly His Gly Gly Asp att tcc Ile Ser 130 atc gaa tat gga acc gat ttg ggc gca cte acc cga ctt gag Ile Glu Tyr Gly Thr Asp Leu Gly Ala Leu Thr Arg Leu Giu aag att cga ctt ttg tee gaa gat ttg gaa age gaa egg gag Lys Ile Arg Leu Leu Ser Glu Asp Leu Glu Ser Glu Arg Glu ega eaa cgt gtt Arg Gin Arg Val ega gaa aag teg gat att ace gtt caa Arg Giu Lys Ser Asp Ile Thr Val Gin 170 ttg atg Leu Met 175 529 aac ttg aee Asn Leu Thr ace gag atg Thr Giu Met 195 cgt ttg gag gag Axg Leu Giu Giu gaa ggt teg agc Giu Gly Ser Ser gaa age gtt Giu Ser Val 190 ctt cga aag Leu Arg Lys aac aag aaa ega Asn Lys Lys Arg tcg gaa ttg get Ser Glu Leu Ala ttg etc Leu Leu 210 gaa gat gte cat Glu Asp Val His gaa age gag gag act get cac cat ttg Giu Ser Giu Giu Thr Ala His His Leu 220 673 eaa aag eac caa Gin Lys His Gin gee att eaa gag Ala Ile Gin Giu caa gat caa ttg Gin Asp Gin Leu cag gtg eaa aag Gin Val Gin Lys aag aae aag teg Lys Asn Lys Ser aag gag aag caa Lys Glu Lys Gin aag ttc Lys Phe 255 WO 01/30817 WO 01/08 17PCT/AUDO/01227 Gin Ala Giu gac aag ttg Asp Lys Leu 275 ri-a gaa ttg ttc* Phe Glu Leu Leu caq gtt gag act Gin Val Glu Thr 9CC aac aag Ala Asn Lys 270 tac acc gta Tyr Thr Val gtc gct cag aag Val Ala Gin Lys gia gaa aag ttg Val Giu Lys Leu cat gag His Giu 290 ttg aac att aag Leu Asn Ile Lys gaa gaa atc aat Giu Giu Ile Asn cga Arg 300 act gtt gtt gag Thr Val Val Giu 913 acc gct cac cga caa cga ttg tcg caa gag aat tcg gaa ttg atc Thr Ala His Arg Gin Arg Leu Ser Gin Glu Asn Ser Giu Leu Ile 310 315 320 aag gaa gtt cat Lys Giu Vai His tac aag att tca Tyr Lys Ile Ser gac aat gct aac Asp Asn Ala Asn cac ttg His Leu 335 1009 aag gga caa Lys Gly Gin gat gaa gaa Asp Giu Glu 355 gca. caa caa ttg Ala Gin Gin Leu gac acc cga cac Asp Thr Arg His cga ttg gag Arg Leu Glu 350 cac aca ttg His Thr Leu 1057 1105 cga aaa cga tca Arg Lys Arg Ser ttg gag aac cac Leu Giu Asn His gaa gtt Giu Val 370 gaa ttg gaa tca Giu Leu Giu Ser aag gta caa ttg Lys Val Gin Leu gag gaa. tct gag Glu Giu Ser Giu 1153 1201 cga tta gaa ttg Arg Leu Giu Leu cga caa ttg act Arg Gin Leu Thr gct aat ggc gat Ala Asn Gly Asp gct tca. tgg aaa tcc aaa tac gaa gcc gaa ctt caa gct cac gtt gat 14 1249 WO 01/30817 WO 0130817PCT/AUOO/01 227 Ala Ser Trp Lys Lys Tyr Giu Ala Leu Gin Ala His Val Asp 415 gaa tat Glu Tyr gag gtt gaa Glu Val Giu ggg gas caa Gly Glu Gin 435 ctt cga cga aag Leu Arg Arg Lys gcc cag sag att Ala Gin Lys Ile tcg Ser 430 1297 1345 ttg gaa gca ttg Leu Glu Ala Leu aac aaa tgt agt gca ttg gas aag Asn Lys Cys Ser Ala Leu Giu Lys caa aag Gin Lys 450 gct cgs ctc caa Ala Arg Leu Gin gag gta gas gtt Glu Val Giu Val stc atg gac ttg Ile Met Asp Leu gaa Giu 465 aag get acc gca Lys Ala Thr Ala gcc csg gca ttg Ala Gin Ala Leu aaa cgc gtt tcc Lys Arg Val Ser 1393 1441 1489 ttg gag sag, ate Leu Giu Lys Ile etc yac etc aag Leu Asp Leu Lys aag ttg gag gag Lys Leu Giu Glu gtc agc Val Ser 495 atg ttg ttg Met Leu Leu ttg eag ass Leu Gin Lys 515 gas Giu 500 caa act cas aag Gin Thr Gin Lys ctc cgt gte sag Leu Arg Val Lys att gcs gac Ile Ala Asp 510 cas sag gas Gin Lys Giu 1537 1585 ttg cas cac gag Leu Gin His Giu gsa sag ttg cga Giu Lys Leu Arg gca ttg Ala Leu 530 gcc cgt gag sac Ala Arg Giu Asn ass etc get gat Lys Leu Ala Asp ttg gct gas get Leu Ala Glu Ala 1633 1681 ass Lys 545 tct cag ttg sat Ser Gin Leu Asn gee cac ega cga Ala His Arg Arg cat gas cas gag His Giu Gin Giu WO 01/30817 WO 0130817PCT/AUOO/01227 -7gag atc aaa cga Glu Ile Lys Arg gag aac gag cga Glu Asn Glu Arg gaa. ttg gct gcc Glu Leu Ala Ala gca tac Ala Tyr 575 1729 aag gaa. gct [Lys Giu Ala ttg act gcc Leu Thr Ala 595 acc ttg cga. aaa Thr Leu Arg Lys gaa gag gca aag Giu Glu Ala Lys aat caa cga Asn Gin Arg 590 aaa cga ttg Lys Arg Leu 1777 1825 gag ttg gca cag Glu Leu Ala Gin cga cac gac tat Arg His Asp Tyr gca caa Ala Gin 610 aag gaa gag gag Lys Giu Giu Giu gaa gct ctt cga.

Glu Ala Leu Arg caa. tac caa att Gin Tyr Gin Ile att gaa caa ttg Ile Giu Gin Leu atg cga. ttg gct Met Arg Leu Ala gct gaa gct aaa Ala Giu Ala Lys 1873 1921 1969 aag acc gag gtt Lys Thr Giu Val cga ctc aag aag Arg Leu Lys Lys aag tac Lys Tyr 650 caa gca. caa Gin Ala Gin atc act Ile Thr 655 gaa. ttg gaa Glu Leu Glu crag aag acg Gin Lys Thr 675 tcg ttg gat gcc Ser Leu Asp Ala aat aag gct aac Asn Lys Ala Asn atc gac ttg Ile Asp Leu 670 ctt caa. gcc Leu Gin Ala 2017 2065 atc aag aag caa.

Ile Lye Lys Gin ctc caa atc act Leu Gin Ile Thr cat tat His Tyr 690 gat gag gta cat Asp Giu Vai His caa ttg caa caa Gin Leu Gin Gin gtt gat caa ttg Val Asp Gin Leu 2113 ggt gtc act caa cga cga tgc caa gca. ttg acc gcc gaa. ttg gag gag 26 2161 WO 01/30817 WO 0130817PCT/AUOO/01227 -8- Gly 705 Val Thr Gin Arg Cys Gin Ala Leu Ala Glu Leu Glu atg cga gte aac Met Arg Val Asn ttg Leu 725 gaa caa gca ctt cgt get aaa cga gct Glu Gin Ala Leu Arg Ala Lys Arg Ala 730 gcc gaa Ala Glu 735 2209 caa atg cac Gin Met His gag gca gtt gtc Glu Ala Val Val gtc aac gaa ctt Val Asn Glu Leu aca ace ate Thr Thr Ile 750 gaa tte tet Giu Phe Ser 2257 aat gte aac ctc get teg gee Asn Val Asn Leu Ala Ser Ala 755 age aaa ttg gag Ser Lys Leu Giu act Thr 765 2305 2353 get ett Ala Leu 770 caa aae gat tac Gln Asn Asp Tyr gaa gta cac aag Giu Val His Lys etc ega ate tet Leu Arg Ile Ser gaa ega gtg caa Giu Arg Val Gin aaa Lys 790 ctt ace att gag Leu Thr Ile Giu aag teg aca aag Lys Ser Thr Lys ttg etc gaa tea Leu Leu Glu Ser aeg gaa egt gte Thr Giu Arg Vai aaa ttg gaa aca Lys Leu Glu Thr ate aag Ile Lys 815 2401 2449 2497 2545 aaa tea ttg Lys Ser Leu gtt gag get Val Giu Ala 835 gaa teg ega Giu Ser Arg 850 gaa aec gaa gta ega aat etc eaa ate ega Glu Thr Giu Val Arg Asn Leu Gin Ile Arg 820 825 aat gca ttg get ggt ggt aaa ega gte atc Asn Ala Leu Ala Gly Gly Lys Arg Val Ile 840 845 att gaa gaa Ile Glu Glu 830 gee aag ttg Ala Lys Leu ate cgt gat Ile Arg Asp gag att gag gtt Glu Ile Giu Val gaa gaa ega ega GIU Giu Arg Arg 2593 WO 01/30817 WO 01/08 17PCT/AUOO/01 227 cac qca qaa act gaa aag atg ctt cga His Ala Glu Thr Giu Lys Met Leu Arg 870 aag gat cat cgt Lys Asp His Arg 2641 aag gaa ttg ttg Lys Glu Leu Leu caa aac gag gag Gin Asn Giu Giu cat aaa caa att His Lys Gin Ile caa ttg Gin Leu 895 2689 ttg caa gaa atg tcc gat aaa ttg Leu Gin Giu Met Ser Asp Lye Leu 900 gaa aag gtc aag Giu Lys Val Lys gtt tac aaa Val Tyr Lys 910 ttg acc cyt Leu Thr Arg 2737 cga caa atg Arg Gin Met 915 caa gaa cag gag Gin Giu Gin Giu atg agc caa caa Met Ser Gin Gin 2785 gtc cga ValI Arg 930 cga ttc caa cga Arg Phe Gin Arg ttg gaa gcc gcc Leu Giu Ala Ala gac cga gct gat Asp Arg Aia Asp caa gct gaa tcc aat Gin Aia Giu Ser Asn 945 gtc acc acc agt caa Val Thr Thr Ser Gin tcg ttc atc cga Ser Phe Ile Arg aaa cat cga tcg Lys His Arg Ser 2833 2881 2929 gtt cct ggc ggt Val Pro Gly Giy cga caa gtc ttt Arg Gin Vai Phe gtc acg Vai Thr 975 gaa caa gag Giu Gin Giu att tca tat Ile Ser Tyr 995 tcg aac ttt Ser Asn Phe taa ata Ile 985 aca gca tgc aac Thr Ala Cys Asn aaa ttc att.

Lys Phe Ile 990 aac ata gat Asn Ile Asp 2977 3025 aaa tcg aac aat aat Lys Ser Asn Aen Asn 1000 ttg aaa ttc aaa cca Leu Lys Phe Lys Pro 1005 ata aat cta ata aaa aaa aag ttt taa aaa aat cac aac ctc tta aac 37 3073 WO 01/30817 PCT/AU00/01227 Ile Asn Leu Ile Lys Lys Lys Phe 1010 1015 aca cct cga tta gcg ttt acc acc act Thr Pro Arg Leu Ala Phe Thr Thr Thr 1025 1030 Lys Asn His Asn Leu Leu Asn 1020 atc acc acc tc Ile Thr Thr 1035 3111 <210> 3 <211> 8 <212> PRT <213> Blomia tropicalis <400> 3 Thr Tyr Ser Asn Gin Thr Leu Val 1 <210> 4 <211> 49 <212> PRT <213> Blomia tropicalis <400> 4 Val Val His 1 Tyr Ile His Gly Val His Ser Ser Ser Ser Ile Arg Pro Asn Asn 5 10 Thr Tyr Thr Leu Thr Thr His Ile Arg 25 Lys Val Ser Gin Pro Val Pro Ser Ser Pro Glu Ala Ile Arg Lys Ser Leu Thr Ser Ser <210> <211> 13 <212> PRT <213> Blomia tropicalis WO 01/30817 PCT/AUOO/01227 11 <400> Thr Ile Asn Glu Ile Ser Leu Ile Leu Leu Leu Lys His 1 5 <210> 6 <211> 14 <212> PRT <213> Blomia tropicalis <400> 6 Thr Pro Phe Tyr Thr Asn Leu Gin Val Tyr Thr Ile Phe Gin 1 5 <210> 7 <211> 4 <212> PRT <213> Blomia tropicalis <400> 7 Asp Ile Arg Glu 1 <210> 8 <211> 3 <212> PRT <213> Blomia tropicalis <400> 8 Asn Gin Tyr 1 <210> 9 <211> <212> PRT WO 01/30817 PCT/AU00/01227 -12- <213> Blomia tropicalis <400> 9 Phe Asp Leu Lys Gin 1 <210> <211> 878 <212> PRT <213> Blomia tropicalis <400> Ser Thr Thr Met Ala Ala Arg Ser Ala Lys Tyr Met Tyr Gin Ser Ser 1 5 10 Arg Ala Gly His Gly Gly Asp Ile Ser Ile Glu Tyr Gly Thr Asp Leu 25 Gly Ala Leu Thr Arg Leu Glu Asp Lys Ile Arg Leu Leu Ser Glu Asp 40 Leu Glu Ser Glu Arg Glu Leu Arg Gin Arg Val Glu Arg Glu Lys Ser 55 Asp Ile Thr Val Gin Leu Met Asn Leu Thr Glu Arg Leu Glu Glu Thr 70 75 Glu Gly Ser Ser Glu Ser Val Thr Glu Met Asn Lys Lys Arg Asp Ser 90 Glu Leu Ala Lys Leu Arg Lys Leu Leu Glu Asp Val His Met Glu Ser 100 105 110 Glu Glu Thr Ala His His Leu Arg Gin Lys His Gin Ala Ala Ile Gin 115 120 125 Glu Met Gin Asp Gin Leu Asp Gin Val Gin Lys Ala Lys Asn Lys Ser 130 135 140 Asp Lys Glu Lys Gln Lys Phe Gin Ala Glu Val Phe Glu Leu Leu Ala 145 150 155 160 Gin Val Glu Thr Ala Asn Lys Asp Lys Leu Val Ala Gin Lys Thr Val 165 170 175 Glu Lys Leu Glu Tyr Thr Val His Glu Leu Asn Ile Lys Ile Glu Glu 180 185 190 Ile Asn Arg Thr Val Val Glu Val Thr Ala His Arg Gin Arg Leu Ser WO 01/30817 PCT/AU00/01227 -13- Gin Leu 225 Asp Glu Gin Thr Glu 305 Ala Lys Glu Leu Ser 385 Leu Lys Ala Arg Glu 465 Glu Glu Leu Asn His 230 Arg Leu 245 His Thr Glu Ser Gly Asp His Val 310 Ser Glu 325 Leu Glu Met Asp Val Ser Glu Val 390 Ile Ala 405 Gin Lys Ala Glu Gin Glu Ala Ala 470 Asn Gln 485 490 495 His Asp Tyr Glu Lys Arg Leu Ala Gin Lys Glu Glu Glu Ile Glu Ala WO 01/30817 WO 0130817PCT/AUOO/01227 14 Gin Leu Asn Met Arg Leu 525 Val Ala Arg Leu Lys Lys 540 Leu Ser LeU Asp Ala Ala 555 560 Ile Lys Lys Gin Ala Leu 575 Glu Val His Arg Gin Leu 590 Gin Arg Arg Cys Gin Ala 605 Asn Leu Giu Gin Ala Leu 620 Giu Glu Ala Val Val Arg 635 640 Leu Ala Ser Ala Lys Ser 655 Asn Asp Tyr Asp Glu Val 670 Val Gin Lys Leu Thr Ile 685 Ser Glu Thr Glu Arg Val 700 Glu Thr Glu Val Arg Asn 715 720 Asn Ala Leu Ala Gly Gly 735 Ile Arg Asp Val Glu Ilie 750 Glu Thr Glu Lys Met Leu 765 Leu Leu Gin Asn Giu Giu 780 Met Ser Asp Lys Leu Aen 795 800 785 790 Glu Lye Val Lys Val Tyr Lye Arg Gin Met Gin Giu Gin Glu Gly Met WO 01/30817 PCT/AU00/01227 805 Gin Gin Asn Leu Thr Arg Val Arg 820 825 Ala Glu Asp Arg Ala Asp Gln Ala 835 840 Ala Lys His Arg Ser Trp Val Thr 850 855 Arg Gin Val Phe Val Thr Glu Gin 870 815 Phe Gin Arg Glu Leu Glu 830 Ser Asn Leu Ser Phe Ile 845 Ser Gin Val Pro Gly Gly 860 Ser Ser Asn Phe 875 <210> 11 <211> 32 <212> PRT <213> Blomia tropicalis <400> 11 Ile Thr Ala Cys Asn Lys Phe Ile Ile Ser Tyr Lys Ser Asn Asn Asn 1 5 10 Leu Lys Phe Lys Pro Asn Ile Asp Ile Asn Leu Ile Lys Lys Lys Phe 25 <210> 12 <211> 19 <212> PRT <213> Blomia tropicalis <400> 12 Lys Asn His Asn Leu Leu Asn Thr Pro Arg Leu Ala Phe Thr Thr Thr 1 5 10 Ile Thr Thr <210> 13 <211> 2625 <212> DNA WO 01/30817 WO 0130817PCT/AUOO/01227 -16- 4213> Biomia tropicalis <220> <221> CDS <222> (1)..(2625) <400> 13 atg geg Met Ala 1 gct cga tea kla Arg Ser 5 gca aag tat atg Ala Lys Tyr Met cag tce agt cgt get gga Gin Ser Ser Arg Ala Gly cac ggc ggc His Gly Gly att tcc ate gaa tat gga ace gat ttg Ile Ser Ile Giu Tyr Gly Thr Asp Le~u ggc gca etc Gly Ala Leu ttg gaa age Leu Giu Ser ace cga ctt gag gac aag att Thr Arg Leu Glu Asp Lys Ile ctt ttg tee gaa Leu Leu Ser Glu gaa egg Glu Arg so gag ctt cga caa Glu Leu Arg Gin gtt gaa cga gaa Val Glu Arg Glu teg gat att ace Ser Asp Ile Thr gtt caa ttg atg aae ttg ace gaa egt ttg Val Gin Leu Met Asn Leu Thr Giu Arg Leu age gaa age gtt ace gag atg aac aag aaa Ser Giu Ser Val Thr Glu Met Asn Lys Lys 90 gag ace gaa ggt Giu Thr Giu Gly ega gac teg gaa Arg Asp Ser Giu ttg get Leu Ala aag ctt ega Lys Leu Arg ttg etc gaa gat gte eat atg gaa age gag gag act Leu Leu Glu Asp Val His Met Glu Ser Giu Glu Thr 105 110 gct cac cat ttg aga caa aag Ala His Hie Leu Arg Gin Lys caa get gee att caa gag atg caa Gin Ala Ala Ile Gin Giu Met Gin 125 WO 01/30817 WO 0130817PCT/AUOO/01227 17 gat caa Asp Gin 130 ttg gat cag gtg Leu Asp Gin Val aag gca aag aac Lys Ala Lys Asn tcg gat aag gag Ser Asp Lys Glu caa aag ttc caa gcc gaa. gtt ttc gaa. ttg ttg gct tag gtt Gin Lys Phe Gin Ala Giu Val Phe Giu Leu Leu Ala Gin Val 480 act gcc aac aag Thr Ala Asn Lys aag ttg gtc gct cag aag acg gta gaa Lys Leu Val Ala Gin Lys Thr Val Glu 170 aag ttg Lys Leu 175 528 gaa. tac acc Giu Tyr Thr cat gag ttg aac att aag att gaa gaa His Glu Leu Asn Ile Lys Ile Giu Giu 185 atc aat cga Ile Asn Arg 190 caa gag aat Gin Glu Asn act gtt gtt Thr Val Val 195 gag gtt acc gct Giu Val Thr Ala cga caa. cga ttg Arg Gin Arg Leu tcg gaa.

Ser Giu 210 ttg atc aag gaa Leu Ilie Lys Giu cat gaa tat aag His Giu Tyr Lys tca ttg gat aat Ser Leu Asp Asn aac cac ttg aag Asn His Leu Lys caa. att gca caa Gin Ile Ala Gin ttg gag gac acc Leu Glu Asp Thr cat cga ttg gag His Axrg Leu Giu gaa gaa cga aaa Giu Giu Arg Lys tca agc ttg gag Ser Ser Leu Giu aac cac Asn His 255 gcc cat aca Ala His Thr gaa. gtt gaa. ttg Giu Val Giu Leu tca ttg aag gta Ser Leu Lys Val.

caa ttg gaas Gin Leu Giu 270 gag gaas ttt gag gcc cga tta gaa ttg gaa cga cas ttg act aaa gct WO 01/30817 WO 0130817PCT/AUOO/01227 18 Glu Giu Ser 275 Giu Ala Arg Leu Leu Giu Arg Gin Thr Lys Ala aat ggc Asn Gly 290 gat get gct tca Asp Ala Ala Ser aaa tee aaa tac Lys Ser Lys Tyr gce gaa cit caa Ala Glu Leu Gin get eae gtt gat gag Ala His Val Asp Giu 305 att tcg gaa tat ggg Ile Ser Giu Tyr Gly 325 gaa gaa ctt ega Glu Glu Leu Arg aag atg gee cag Lys Met Ala Gin 912 960 1008 gaa eaa ttg gaa Glu Gin Leu Giu ttg ttg aac aaa Leu Leu Asn Lys tgt agt Cys Ser 335 gea ttg gaa Ala Leu Giu ate atg gac Ile Met Asp 355 caa aag get ega Gin Lys Ala Arg caa teg gag gta Gin Ser Giu Val gaa gtt ttg Glu Val Leu 350 ttg gag aaa Leu Giu Lys 1056 1104 ttg gaa aag get Leu Glu Lys Ala gca eat gee eag Ala His Ala Gin ege gtt Arg Val1 370 tee eaa ttg gag Ser Gin Leu Giu ate aae etc gae Ile Asn Leu Asp aag age aag ttg Lys Ser Lys Leu gag Glu 385 gag gte age atg Glu Val Ser Met ttg gaa caa act Leu Giu Gin Thr aag gat etc egt Lys Asp Leu Arg 1152 1200 1248 aag att gca gac Lys Ile Ala Asp cag aaa ttg caa Gin Lys Leu Gin gag tac gaa aag Giu Tyr Giu Lys ttg ega Leu Arg 415 gac caa aag Asp Gin Lys gca ttg gee egt Ala Leu Ala Arg gag Glu 425 aac aag aaa etc Asn Lys Lys Leu get gat gat Ala Asp Asp 430 1296 WO 01/30817 WO 01/08 17PCT/AUOO/01227 ttg gct gaa Leu Ala Giu 435 gct aaa tet cag ttg aat gac gcc cac cga cga atc cat Ala Lys Ser Gin Leu Asn Asp Ala His Arg Arg Ile His 1344 gaa caa Glu Gin 450 gag ate gag ate Glu Ile Glu Ile ega ttg gag aac Arg Leu Glu Asn cga gaa gaa ttg Arg Glu Giu Leu 1392 gcc gca tac aag Ala Ala Tyr Lys get gaa acc ttg cga aaa eaa gaa gag Ala Glu Thr Leu Arg Lys Gin Glu Giu 475 1440 aag aat caa cga Lys Asn Gin Arg act gcc gag ttg Thr Ala Giu Leu cag aca cga cac Gin Thr Arg His gac tat Asp Tyr 495 1488 gag aaa ega Glu Lys Arg gca caa aag gaa Ala Gin Lys Glu gag att gaa gct Glu Ile Glu Ala ctt ega aaa Leu Arg Lys 510 1536 caa tac aa Gin Tyr Gin 515 att gaa att gaa caa ttg aac atg ega ttg get gag get Ile Glu Ile Giu Gin Leu Asn Met Arg Leu Ala Giu Ala 1584 gaa get Glu Ala aaa ttg aag ace Lys Leu Lys Thr gtt gea cga etc Val Ala Arg Leu aag aag tac caa Lys Lys Tyr Gin gca caa ate act gaa Ala Gin Ile Thr Giu 545 t tg Leu 550 gaa ttg teg ttg Giu Leu Ser Leu gee get aat aag Ala Ala Asn Lys 1632 1680 1728 aac ate gac ttg Asn Ile Asp Leu aag acg ate aag Lys Thr Ile Lys caa get etc caa Gin Ala Leu Gin ate act Ile Thr 575 ggg ett eaa gee cat tat gat gag gta cat cgt caa ttg caa caa get i7 1776 WO 01/30817 WO 0130817PCT/AUOO/01227 20 Gly Leu Gin Ala His Tyr Asp Giu 580 His Arg Gin Leu Gin Gin Ala 590 ttg ace gcc Leu Thr Ala gtt gat Val Asp ttg ggt gtc act Leu Gly Val Thr cga cga tgc caa Arg Arg Cys Gin 1824 gaa ttg Glu Leu 610 gag gag atg cga Giu Glu Met Arg aae ttg gaa caa Asn Leu Glu Gin ctt egt get aaa Leu Arg Ala Lys 1872 1920 get gec gaa eaa Ala Ala Giu Gin cac gaa gag gca His Giu Giu Ala gtc ega gte aac Val Arg Val Asn ctt aca ace atc aat gte aac etc get Leu Thr Thr Ile Asn Val Asn Leu Ala 645 gec aag agc aaa Ala Lys Ser Lys ttg gag Leu Giu 655 1968 act gaa ttc Thr Glu Phe get ctt caa aac Ala Leu Gin Asn tac gat gaa gta cac aag gaa Tyr Asp Giu Val His Lys Giu 670 2016 etc cga Leu Arg tet gac gaa ega gtg eaa aaa ctt ace Ser Asp Giu Arg Val Gin Lys Leu Thr 680 gag gte aag Giu Val Lys 2064 teg aca Ser Thr 690 aag gae ttg etc Lys Asp Leu Leu tea gaa aeg gaa Ser Giu Thr Glu gte aca aaa ttg Vai Thr Lys Leu aca ate aag aaa Thr Ile Lys Lys ttg gaa ace gaa Leu Giu Thr Giu ega aat etc eaa Arg, Asn Leu Gin 2112 2160 2208 cga att gaa gaa Arg Ile Giu Giu gtt gag Val Giu 725 get aat gca Ala Asn Ala ttg tjLe 730 get ggt ggt aaa Ala Gly Gly Lys ega gte Arg Val 735 WO 01/30817 WO 0130817PCT/AUOO/01227 -21atc qcc aaci Ile Ala Lys gaa gaa cga Glu Glu Arg 755 qaa tcg cqa atc Giu Ser Arg Ile gat gtt gag Asp Val Giu att gag gtt gaa Ile Glu Vai Giu 750 ctt cga aag aag Leu Arg Lys Lys 765 2256 2304 cga cga cac gca Arg Arg His Ala act gaa aag atg Thr GlU Lys Met gat cat Asp His 770 cgt gtc aag gaa Arg Val Lys Glu~ ttg ctt caa aac gag gag gac cat aaa Leu Leu Gin Asn Glu Giu Asp His Lys 780 2352 att caa ttg ttg Ile Gin Leu Leu gaa atg tcc gat Glu Met Ser Asp ttg aac gaa. aag Leu Asn Glu Lys 2400 2448 aag gtt tac aaa Lys Vai Tyr Lys caa atg caa gaa Gin Met Gin Glu gag gga atg agc Giu Gly Met Ser caa caa Gin Gin 815 aac ttg acc Asn Leu Thr gac cga gct Asp Arg Ala 835 gtc cga. cga ttc Vai Arg Arg Phe cga gag ttg gaa Arg Giu Leu Glu gcc gcc gaa Ala Ala Giu 830 cga. gcc aaa Arg Ala Lys 2496 2544 gat caa gct gaa Asp Gin Ala Glu aat ttg tcg ttc Asn Leu 5cr Phe cat cga His Arg 850 tcg tgg gtc acc 5cr Trp Val Thr agt caa gtt cct ggc ggt act cg4 caa Ser Gin Val Pro Giy Gly Thr Arg Gin 860 2592 ttt gtc acg gaa Phe Val Thr Glu gag agc tcg aac Glu Ser Ser Asn 2625 WO 01/30817 WO 0130817PCT/AUOO/01227 22 4210> 14 <211> 875 <212> PRT <213> Blomia tropicalis <400> 14 Met Ala Ala Arg Ser Ala Lys Tyr Met Tyr Gin Ser Ser Arg Ala Gly Ala Leu His Gly Gly Thr Arg Leu Glu Arg Glu Ile Ser Ile Glu Tyr Gly Thr Asp Leu Asp Lys Ile Leu Leu Ser Glu Leu Glu Ser Asp Ile Thr Leu Arg Gin Val Glu Arg Glu Gin Leu Met Ann Giu Arg Leu Thr Glu Gly Giu Scr Val Met Ann Lys Asp Ser Giu Leu Ala Lys Leu Arg Ala His His 115 Asp Gin Leu Leu Giu Asp Met Glu Ser Arg Gin Lys Ala Ala Ile Glu Giu Thr 110 Giu Met Gin Asp Lys Giu Asp Gin Val Ala Lys Ann 130 Lys Gin Lys 140 Leu Lys Phe Gin Val Phe Glu Ala Gin Val Ala Ann Lys Asp 165 Leu Val Ala Thr Val Glu WO 01/30817 WO 0130817PCT/AUOO/01227 23 (Gli Tyrr 'Thr Thr Val Val 195 Ser Giu Leu His Glu Leu Asn Lys Ile Giu Giu Ile Asn Arg 190 Val Thr Ala Gin Arg Leu Gin Giu Asn Leu Asp Asn Ile Lys Giu Giu Tyr Lys 210 Ala Asn His Leu Lys Ile Ala Gin Glu Asp Thr Arg Leu Glu Giu Arg Lys Ser Leu Giu Asn His 255 Ala His Thr Giu Giu Ser 275 Asn Gly Asp Val Giu Leu Leu Lys Val Ala Arg Leu Glu Glu Arg Gin Gin Leu Glu 270 Thr Lys Ala Glu Leu Gin Ala Ala Ser Lys Ser Lys Tyr Giu 290 Ala His Val Asp Giu Giu Leu Arg Lys Met Ala Gin Ser Giu Tyr Gin Leu Glu Ala Leu Asn Ala Leu Giu Ile Met Asp 355 Arg Val Ser Lys Ala Arg Gin Ser Giu Val Lys Cys Ser 335 GiU V/al Leu 350 Leu Giu Lys Ser Lys Leu Giu Lys Ala His Ala Gin Gin Leu Glu Lys Asn Leu Asp Leu WO 01/30817 PCT/AU00/01227 -24- Val Ser Met Glu Gin Thr Asp Leu Arg Ile Ala Asp Lys Leu Gin Tyr Glu Lys Leu Arg 415 Asp Gin Lys Leu Ala Glu 435 Glu Gin Glu Leu Ala Arg Lys Lys Leu Lys Ser Gin Asp Ala His Ala Asp Asp 430 Arg Ile His Glu Glu Leu Ile Glu Ile Leu Glu Asn 450 Ala Ala Ala Tyr Lys Asn Gln Arg Thr Ala Glu Thr Leu Glu Leu Ala 490 Glu Glu Glu Gin Glu Glu Thr Arg His Asp Tyr 495 Glu Lys Arg Gin Tyr Gin 515 Glu Ala Lys Gin Lys Ile Glu Ala Glu Ile Glu Asn Met Arg Leu Arg Lys 510 Ala Glu Ala Lys Tyr Gin Leu Lys Thr Ala Arg Leu 530 Ala Gin Ile Thr Glu Leu Ser Leu Ala Asn Lys Ile Asp Leu Thr Ile Lys Ala Leu Gin WO 01/30817 WO 0130817PCT/AUOO/01 227 25 Gly Leu Gin His Tyr Asp Giu His Arg Gin Leu Gin Gin Ala 590 Leu Thr Ala Val Asp Gin 595 Leu Gly Val Thr Gin Arg Arg Cys Gin 600 Glu Leu 610 Gbli Giu Met Arg Asn Leu Glu Gin Ala Leu Arg Ala Lys 620 Val Arg Val Asn Glu Ala Ala Giu Gin His Giu Giu Ala Leu Thr Thr Ile Val Asn Leu Ala Ser 650 Ala Lys Ser Lys Leu Glu 655 Thr Giu Phe Leu Arg Ile 675 Ala Leu Gin Asn Tyr Asp Giu Val His Lys Giu 670 Giu Val Lys Ser Asp Giu Arg Gin Lys Leu Thr Ser Thr 690 Lys Asp Leu Leu Ser Giu Thr Glu Val Thr Lys Leu Glu 705 Thr Ile Lys Lys Leu Giu Thr Giu Arg Aen Leu Gin Arg Ilie Giu Glu Glu Ala Aon Ala Ala Gly Gly Lys Arg Val 735 Ile Ala Lys Giu Giu Arg 755 Giu Ser Arg Ile Asp Val Giu Ile Giu Val Glu 750 Arg Lys Lys Arg Arg His Ala Thr Giu Lys Met Asp His 770 Arg Val Lys Giu LeU Leu Gin Asn Giu Asp His Lys WO 01/30817 WO 0130817PCT/AUGO/01227 26 ie Glin T.eu Leui Ciu Met Ser AsD Leu Asn Glu Lys Lys Val Tyr Lys Gin Met Gin Giu Glu Gly Met Ser Gin Gin 815 Asn Leu Thr Asp Arg Ala 835 Val Arg Arg Phe Arg Giu Leu Glu Ala Ala Giu 830 Arg Ala Lys Asp Gin Ala Giu Asn Leu Ser Phe His Arg 850 Ser Trp Val Thr Ser Gin Val Pro Gly Thr Arg Gin Phe Val Thr Glu Glu Ser Ser Asn <210> iS <211> 701 <212> PRT <213> Dermatophagoides farinae <400> Met Asn Lys Lys Arg Asp Ser Giu Leu Arg Lys Leu Arg Lys Leu Leu is Glu Asp Val Ile Glu Ser Glu Thr Ala His His Leu Arg Gin Lys His Gin Ala Ala Ile Gin Met Gin Asp Gin Leu Asp Gin Leu Gin Lys Ala Lys Asn Lys s y i y y h i l Asp Lys Glu Lys Lys Phe Gln Ala WO 01/30817 PCT/AU00/01227 -27- Val Phe Glu Leii Ala fin Leu Glu Ala Asn Lys Glu Leu Thr Ala Leu Asn Val Glu Lys Leu Glu Tyr Thr Val His Glu Leu Asn Ile lie Glu Glu Ile Arg Thr Val Ile Glu Leu Thr.

110 Ser His Lys 115 Gin Arg Leu Ser Gln Glu Asn Thr Glu Leu Ile Lys Glu Val His Glu Val Lys Leu 130 Leu Asp Asn Ala His Leu Lys Thr Ile Ala Gin Gin Glu Asp Thr Arg Arg Leu Glu Glu Glu 160 Glu Arg Lys Arg Ser Leu Glu Asn His Ala His Thr Leu 170 Glu Val 175 Glu Leu Glu Ser Leu Lys Val Gin Leu Asp Glu Glu Ser 180 185 Glu Ala Arg 190 Ala Ala Ser Leu Glu Leu 195 Glu Arg Gin Leu Thr Lys Ala Asn Gly 200 Trp Lys Ser Lys Tyr Glu 210 Glu Leu Gin Ala His Glu Asp Glu Val 220 Ser Glu Tyr Glu Glu 240 Glu Leu Arg Arg Lys Met Ala Gin Lys 230 Gin Leu Glu Ala Leu Asn Lys Cys Ser Leu Glu Lys Gin Lys 255 Ser Arg Leu Gin Ser Glu Val Glu Val Leu Ile Met Asp Leu Glu Lys WO 01/30817 PCT/AU00/01227 -28- Ala Thr Arg 275 Ala Gin Gin Lys Arg Val 270 Gin Leu Glu Thr Met Leu Lys Ile 290 Met Glu 305 Lys Leu Asn Leu Asp Leu Lys Leu Glu Gin Ala Gin Lys Leu Arg Val Ala Glu Leu Gin His Tyr Glu Lys Leu Gin Arg His Gin Leu 335 Ala Arg Glu Gin Leu Asn 355 Lvs Arg Leu Lys Leu Thr Leu Ala Glu Ala His Arg His Glu Gln Ala Lys Ser 350 Ile Glu Ile Tyr Lys Glu Glu Asn Glu Glu Leu Ser 370 Ala Glu Thr Leu Arg Glu Glu Ala Gin Arg Leu Glu Leu Ala Arg His Asp Lys Arg Leu Ala Gin 415 Lys Asp Glu Glu Gin Leu 435 Glu Ile Ala 450 Glu Ala Leu Gin Tyr Gin Asn Met Arg Leu Arg Leu Lys Lys 455 Ala Glu Ala Lys 445 Ile Ile Glu Ile 430 Leu Lys Thr Thr Glu Leu Tyr Gin Ala WO 01/30817 WO 0130817PCT/AUO/01227 29 Leu Ser Leu Asp Ala Asn Lys Ala Ile Asp Leu Gin Thr Met Lys Lys Gin Ala Leu Gin Ile Thr Giu Leu Gin Ala His Tyr 495 Asp Glu Val His Arg Gin Leu Gin Ala Vai Asp Gin Leu Giy Val 510 Giu Met Arg Thr Gin Arg 515 Arg Cys Gin Ala Gin Ala Glu Leu Ile Ala Leu Giu Gin Ala 530 Arg Ala Lys Ala Ala Glu Gin Leu Giu Giu Ala Val Arg Val Asn Giu Thr Thr Ilie Asn Val 560 Asn Leu Ala Ser Ala Lys Ser Lys Leu Giu Ser Giu Phe Ser Ala Leu 575 565 570 Gin Ala Asp Tyr Asp Giu Val HiB 580 Arg Vai Gin Lys Leu Thr Ilie Glu 595 600 Giu Leu Arg Ile Ser Asp Giu 590 Asp Leu Leu Leu Lys Ser Thr Ile Giu Glu Gin Giu Arg 610 Vai Lys Leu Giu Val Lys Lys Ser Giu Gin Giu Val Arg 630 Thr Leu His Val Ile Giu Giu Val Ala Asn Ala Leu Ala Gly Gly Lys Arg 645 Ile Ala Lys Leu Giu Ser 655 Arg Ile Arg Val Glu Ile Giu Giu Glu Glu Arg Arg Arg His 670 WO 01/30817 WO 0130817PCT/AUOO/0 1227 Ala Glu Thr Asp Lys Met Leu Ala Lys 675 680 Leu Leu Leu Gin Pro Pro Glu Arg Glu 690 695 Lys Asp His Arg Val Lys Glu 685 His Pro Arg Val 700 <210> 16 <211> 878 <212> PRT <213> Drosophila melanogaster <400> 16 Met Ser Ser Ser Gin Ala Val Arg Ser 1 5 Lys Tyr Ser Tyr Arg Ala Thr Ser Thr Pro Gly Thr Ala Asp Val Asn Ile Giu Tyr Ile Gin Leu Leu Gin Asp Leu Ser Ser Leu Ser Arg Glu Asp Lys Ile Asp Asp Leu Giu Val Glu Glu Leu Arg Gin Ile Giu Arg Giu Lys Ala Asp Leu Ser Gin Val Ile Gin Ser Giu Arg Leu Giu Ala Giu Giy Ala Glu His Gin Glu Ala Asn Arg Lys Arg Asp Ala Glu Leu Lys Leu Arg Leu Leu Glu Asp Val His Leu 110 Giu Ser Giu 11s Giu Thr Thr Leu Leu Lys Lys Lys His Asn Glu Ile WO 01/30817 PCT/AU00/01227 -31 Ile Thr 130 Arg Ala Asp Phe Gin Glu Val Glu Ile Leu Lys Asn Lys Ala Glu Lys Asp Lys Phe Gin Val Tyr Glu Ser Gin Ile Tyr Aen Lys His Ile Ser Glu Glu Leu 195 Leu Ser Gin Glu Val Ser Glu Lys Ile 170 Ser Glu Leu Ile Ser Ser Val Ser Asn Arg Thr Val Glu Lys 175 Val Lys Ile 190 Arg Ser Arg Asp Leu Lys Glu Asn Ile Thr Lys Asp Val 225 Leu Leu Asp Thr Phe Ser Lys Val Ile Ser Gin 240 Glu Asp Ala Arg Arg Leu Glu Leu Leu Glu Arg Asn Gin 275 Gin Leu Val Ser Leu His Gln Asp Glu Asp 250 Glu Ile Glu Ala Arg Ile Arg Arg Arg Ser 255 Leu Glu Glu Glu Asp Ser val 270 Leu Glu Arg Asn Lys Trp Lys Ala Asn Ala Thr Ser 290 Asn Ser 305 Glu Val Ala Ala Glu Glu Glu Ile Arg Lys Tyr Gin Val Arg Ile Thr Glu Leu Glu Glu His Ile Glu Ser Leu WO 01/30817 WO 0130817PCT/AUOO/0 1227 -32- Ile Val Lys Giu Val Glu 355 Arg Giu Leu 370 Leu Lys Ser 385 Arg Asp Leu Leu Asp Lys Lys Leu Gly 435 Asn Arg Arg Asn Asn Leu Giu Met Lys Thr Arg Leu Ile Ile Glu Lys Ser Asn 365 His Leu Ala Ser 350 Asn Ser Cys Asn Val Giu Thr Lys Ser Thr Leu Glu Arg Leu Lys Asn 405 Val Lys Thr Ile Ile Glu Thr Ser His Ala Asp Leu Arg Thr Asp Asn Asn Gin Leu Thr Arg Val His Giu 415 Glu Asn Lys 430 Asn Glu Leu Leu Glu Asn Asp Leu His Lys Gly Aia 450 Arg Asp Ala Giu Leu His Glu Giu Lieu Thr 470 Giu Gin Arg Leu Giu Leu Arg 460 Ala Ala Tyr Lys Giu Ala Gly Gly Gin Arg Ala Asp Phe Asn Gin 495 Tyr Arg His Ala Ile Arg 515 Val Ile Giu Arg Arg Leu Lys Asp Giu Gin Thr Ser Ile Giu Gin Giu Ile Glu 510 Asn Ala Arg Arg Ile Lys Ala Giu Thr Arg Lys Thr Glu Val WO 01/30817 WO 0130817PCT/AUOO/01227 33 530 Lys Leu Gin Ile Gin 550 Thr Asn Ile Thr Giu Leu Ser Leu Asp Asn Lys Asp Leu Gin Ile Lys Lys Gin Ser 575 Leu Gin Leu Leu Gin Ala 595 Giv Leu Asn Leu Gin Ala Giu Asp Val Leu Asp Gin Val Ala Gin Gin Arg Gin 590 Arg Leu Ala Asp Ser Aia Gly Giu Leu Val Arg Ser 610 Asn Arg Ala Lys Arg Glu Leu Gin Giu Ala Ala Ile Asn Glu Thr Ala Asn Leu Val Ser Ile Lys 655 Ser Lys Leu Val Ser Lys 675 Vai Giu Leu Giu Leu Ser Ala Ser Asp Leu Arg Ile Giu Arg Tyr Tyr Giu Giu 670 Lys Val Gin Gin Giu Arg Lys His Vai Gin Val His 690 Ile Val Lys Leu Giu Lys Lys Ser Val Glu Val Leu Ser IlieGi uVae AlVi Glu Glu Val Asn Ala Val WO 01/30817 PCT/AU00/01227 -34- Ser Lys Arg Leu Glu Leu 755 Ile Ser Lys Leu Ala Arg Ile Arg Asp Leu Glu 750 Ile Lys lie Glu Glu Glu Lys Arg His Ala Glu Leu Arg 770 Lys Lys Glu Arg Val Lys Glu Val Leu Val Gin Cys Glu 780 Asp Gin Lys Asn Ile Leu Leu Gin Ala Leu Asp Lys Thr Ala Lys Ile Ile Tyr Arg Arg Leu Ser Glu Gin Glu Gly 815 Val Ser Gin Thr Thr Thr Arg Arg Arg Phe Gin Arg Glu Leu 830 Leu Asn Ile Glu Ala Ala 835 Glu Asp Arg Ala Asp Thr Ala Glu Ser 840 Ile Arg 850 Ala Lys His Arg Thr Phe Val Thr Thr 855 Thr Val Pro Gly Ser Gin Val Tyr Ile 865 Glu Thr Thr Arg Ile Thr Glu <210> 17 <211> 891 <212> PRT <213> Onchocerca volvulus <400> 17 Met Ser Gly Ser Leu Tyr Arg Ser Pro Ser Ala Ala Leu Tyr Lys Ser 1 5 10 Pro Ser Met Ser Ala Phe Ser Gly Leu Pro Ala Ala Phe Gly Ser Met WO 01/30817 PCT/AU00/01227 Ser Val Ala Leu Leu Gln Glu Arg Glu Leu Gly Ser Arg Leu Glu Lys Ile Arg Asn Arg Ile Glu Asp Leu Glu Glu Arg Glu Arg Ala Leu Ser Val Gin Ala Leu Thr Leu Glu Asp Gly Thr Thr Gin Ile Glu Ser Asn Arg Lys Arg Ser Gin Leu 115 Gin Asp Ala Glu Leu Gin Arg Lys Leu Asn Glu Asp Asn Val Leu Leu Glu Glu 110 Lys Lye His Leu Gin Lys Cys Leu Asp Glu Gin Ile 130 Lvs Asn Ser Lys Ile Glu Arg Gin Gin His Glu Glu Leu Thr lie Asp Gin Lys Asp Lys His Leu 175 Ala Glu Lys Asn Lys Val 195 Glu Arg Phe Gin Thr Ile Glu Leu Ser 190 Ala Gin Gin Asp Leu Asn Val Asn Asp Arg Gin Arg Leu Gin Ala Glu Asn Asn Asp Leu Leu Lys Glu Ile His 210 215 220 WO 01/30817 WO 0130817PCT/AUOO/01227 -36- Gin Lys Val Gin Asp Asn Leu Gin Val Lys Tyr Gin Ala Gin Gin Leu Giu Ala Arg Arg Leu Giu Asp Ala Glu Arg 255 GiU Arg Ser Asp Ser Val 275 Leu Gin Ala Gin His Gin Val Gin Leu Giu Leu 270 Arg Ala Giu Arg Thr Ala Leu Giu Glu Ser Ala Ala Giu 290 His Lys Leu Ala Ala Asn Thr Giu Thr Gin Trp Lys Lys Phe Asp Ala Val Ala Leu His Glu Glu Val Glu Asp 320 Leu Arg Lys Lys Leu Gin Lys Gin Al a 330 Glu Tyr Giu Giu Gin Ile 335 Giu Ile Met Leu Gin Ser 355 Gin Lys Ile Ser Leu Giu Lys Ala Lys Ser Arg 350 Lys Ala Gin Giu Val Giu Val Ile Val Asp Leu Asn Thr 370 Ile Ala Ile Leu Giu Arg Ala Lys Giu 375 Leu Giu Lys Thr Met Giu Leu Lys Arg Ile Asp Glu Thr Val Giu Leu Ala Ala Gin Arg Ala Arg Ala Ala Ala Glu Leu Gin Lys Met 415 Lys Asn Leu Tyr Giu 420 Lys Ala Val Giu Gin Lys Giu Ala Leu Ala Arg WO 01/30817 WO 0130817PCT/AUDO/01227 -37- Giu Asn Lys 435 Ala Asp Ala Lys Leu Gin Asp Leu His Giu Ala Glu Ala Leu Asn Ala Arg Asn Arg Lys Glu Leu Asp 450 Leu Ala Gly Giu Ile Leu Gin Thr Lys Glu Ser Ala Arg Arg Ala Giu Asn Arg Arg Ala Leu Ala Giu 495 Leu Gin Gin Glu Giu Met 515 Leu Thr Ala Ile Giu Met Arg Leu Gin Ala Leu Arg Met Gin Phe Glu Lys Giu 510 Ile Asp Arg Ala Giu Ile Ala Leu Ala Giu Ala Arg 530 Ala Arg 545 Thr Val Leu Lys Lys Lys Gin Ala Glu Giu Leu Glu Asp Asn Asn Arg Ala Asfl Ala Gin Lys Thr Ile 575 Lys Lys Gin Thr Gin Arg 595 Arg Lys Val Ser 580 Gin Gin Leu Lys Gin Ala Ser Leu Gin Gin LeU Asp Gin Tyr Leu Glu Asp 590 Leu Ala Gin Lys Val Ala Ser Ala Leu Giu Leu Glu 610 Leu Asp Asn Ala Ile Arg Ann Aa li Arg Arg Lys Gin Ala leAsLuGi Ile Asp Leu Glu WO 01/30817 PCT/AU00/01227 -38- Ala Asn Gly Thr Asp Leu Ile Asn Asn 640 Asn Leu 655 Thr Ala Ile Asp Leu Asp 675 Asn Arg Ala Lys Leu Glu Leu Ser Thr Ala Thr Lys His Ala Ala Asp 685 Gin Ala Gin Ala 670 Glu Arg Ala Leu His Glu Leu Ala Asp Arg Ala Val 690 Glu Gin Glu His Ser Ile Asp Ala Lys Ser Leu 705 Glu Gin Val Lys Gin Val Gin Glu Ala Glu Ala Ala 735 Ala Leu Leu Arg Asp Leu 755 Thr Gin Gly Gly Lys Arg Val Lys Leu Glu Thr Ala Leu Glu Thr Arg Thr Arg Ile 750 His Lys Glu Glu Val Gin Ala Leu Arg Asp Arg Arg 770 Met Gin Val Asp Glu Lys Met Phe Ala Gin Asp 785 Ala Asp Arg Leu Lys Leu Asn Lys Arg Gin Leu Gly 815 Arg Tyr Glu Ala Glu Thr Met Ala Gin Arg Val WO 01/30817 PCT/AUOO/01227 -39- Gin Arg Giu Leu Giu Asp Ala Glu Asp Arg Ala Asp Thr Ala Glu Ser Ser Leu 850 An Ilie Ile Arg Lys His Arg Thr Phe Val Thr Thr Ser 860 Thr Val Pro Gly Ser 865 Thr Giu Pro Pro Giu Val Tyr Ile Gin Thr Thr Arg Thr Arg Glu His Pro Arg Vai1 890 <210> 18 <211> 872 <212> PRT <213> Caenorhabditis elegans <400> 18 Met Ser Leu Tyr Arg 1 5 Ser Pro Ser Ala Ala Leu Leu Lys Ser 10 Pro Ser is Gin Ala Ala Phe Gly Ala Pro Phe Gly Ser Met Ser Val Ala Asp Leu Gin Glu Asp Gly Ser Leu Thr Arg Leu Glu Asp Lys Ile Arg Leu Leu Glu Ser Giu Arg Giu Arg Asn Arg Val Axg Giu Arg Ala Asp Leu Ser Val Gln Ile Ala Leu Thr Arg Leu Glu Asp Glu Gly Thr Thr Ser Gin Ile Glu Asn Arg Lys Arg Giu Gly Giu Leu Ser Lys Leu Arg Lys Leu Leu Giu Giu Ser Gin Leu Giu Ser WO 01/30817 WO 0130817PCT/AUOO/01227 40 Glu Asp Ala 11.5 Asp Tyr Gin Asn Val Leu Lys His Gin 110 Ser Cys Leu Ala Lys Ile Asp Gin Ile Leu Gin Lys Glu Arg Gin Gin His Giu Giu Leu Thr Ile Asp Gin Lys Asp Lys Ala Giu Lys Ala Ala 175 Giu Arg Phe Leu Asn Lys 195 Ala Glu Asn Gin Ala Asn Ala Asn Lys Val Asn Asp Gin Gin Arg Val Giu Asp 190 Arg Leu Gin Lys Vai Gin Asn Asp Leu Glu Val His 210 Leu Asp Asn Leu Gin Lys Tyr Thr Gin Gin Leu 225 Glu Ala Arg Arg Arg 245 His Giu Asp Ala Giu Arg Ser Gin Leu 255 Gin Ser Gin Ala Leu Asp 275 Asn Leu Ala 290 Gin Val Gin Leu Asp Ser Glu Ser Ile Ser Asp Ala Val Arg Thr 270 His Lys Leu Phe Asp Ala Asn Thr Glu Gin Trp Lys WO 01/30817 WO 0130817PCT/AUOO/01227 -41- Giu 305 Leu Val Ala Leu His Glu Giu Val Giu Leu Arg Lys Lys Gin Lys Gin Tyr Giu Glu Giu Ile Met Leu Gin 335 Lys Ile Ser Glu Val Leu 355 Leu Giu Arg Glu Lys Ala Arg Leu Gin Val Asp Leu Ala Gin Asn Thr 365 Gly Ser GiU Val 350 Ile Ala Leu Giu Leu Lys Ala Arg Giu Giu Arg Gin 370 Val Arg Ile Asp Giu Val Giu Leu Ala Gin Arg Arg Ala Val Glu Leu Gin Lys His Leu Tyr Giu 415 Lys Ala Val His Asp Giu 435 Lys Leu His Lys Glu Ala Arg Glu Asn His Glu Ala Ala Leu Ala Lys Lys Leu 430 Ala Asn Arg Giy Giu Ile Glu Leu Asp Aen Ala Arg Leu Gin Thr Lys Giu Ala Asp 475 Giu Gin Arg Arg Giu Asn Arg Arg Ala Leu Leu Gin Ala Leu Arg 495 Glu Ala Ile Glu Met Arg Leu Gin Glu 505 Giu Giu Giu met 510 WO 01/30817 WO 0130817PCT/AUOO/0 1227 -42 Leu Arg Lys Asn Leu Gin Phe Giu Ile Asp Arg Leu Ala Ala Leu Ala Asp 530 Ala Glu Ala Arg Lys Ser Glu Ile Ser Arg Leu Lys Lys 540 Tyr Gin Ala Giu Ala Glu Leu Giu Met Thr Val Asp 555 Ile Lys Lys Gin As n Asn Arg Ala Asn Giu Ala Gin Lys Ser Giu 575 Gin Leu Lys Ile Leu Gin Ala Ser 580 Giu Asp Thr Gin Arg Gin Leu 590 Gin Gin Val Leu Asp Gin Tyr Ala Leu Ala Gin Arg Lys Val Ala Ala 600 605 Leu Ser 610 Ala Giu Leu Glu Cys Lys Thr Ala Asp Asn Ala Ile Ala Arg Lys Gin Ala Glu Val Asp Leu 630 Glu Ala Asn Gly Ile Ser Asp Leu Ser Ile Asn Asn Leu Thr Ser Ilie Lys Aen 655 Lys Leu Giu Thr Lys Glu 675 Giu Leu Ser Thr Gin Ala Asp Leu Asp Glu Val 670 Ala Leu Ala Leu His Ala Ala Asp 680 Giu Arg Ala Aen Asp Ala 690 Ala Arg Ala Val Giu Gin Leu His Glu Giu Gin Glu His Ser 695 700 Met Lys Ile Asp Ala Leu Arg Lys Ser Leu Giu Giu Gin Val Lye Gin WO 01/30817 WOO1/0817PCT/AUOO/01227 -43- 720 Gly Gly 735 Leu Gin Val Gin Gin Glu Ala Giu Ala Ala Ala Leu Leu 730 Lys Arg Val Ala Leu Asp 755 Ala Lys Leu Glu Arg Ile Arg Asp Leu Glu Thr 750 Asn Ala Leu Glu Giu Thr Arg His Lys Glu Thr Arg Lys 770 Lys Asp Arg Arg Lys Giu Val Gin Leu Val Asp Glu His Lys Asn Phe Met Ala Gin Asp Ala Asp Arg Leu Glu Lys Leu Asn Gin Lys Arg Gin Ala Glu Ser Glu Ser Val 815 Thr Met Gin Asp Ala Giu 835 Leu Gin Arg Val Arg Tyr Gin His Giu Leu Giu 830 His Leu Ile Gly Arg Ala Asp Ala Glu Ser Ser Arg Ala 850 Lys His Arg Ser Val Val Thr Gly Lys 860 Ser Ser Ser Lys Phe Val Thr Giu Asp Asp Tyr 870 <210> 19 <211> <212> PRT <213> Artificial Sequence WO 01/30817 WO 0130817PCT/AUOO/0 1227 44- <220> <223> Description of Artificial Sequence:primer <400> 19 Ala Ala Thr 1 Thr Ala Ala Cys Cys Cys Thr Cys Ala Cys Thr Ala Ala 5 10 Ala Gly Gly Gly <210> <211> <212> <213> <220> <223> 22

PRT

Artificial Sequence Description of Artificial Sequence:primer <400> Cys Gly Gly

I

Gly Ala Thr Ala Thr Cys Ala Cys Thr Cys Ala Gly Cys 5 10 Ala Thr Ala Ala Thr Gly <210> 21 <211> <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence~prinler <400> 21 Cys Thr Thr Cys Ala Thr Cys Cys Thr Cys Cys Ala Ala Thr Cys Gly 1 5 10 WO 01/30817 PCT/AU00/01227 Gly Thr Gly Thr <210> <211> <212> <213> <220> <223> 22

PRT

Artificial Sequence Description of Artificial Sequence:primer <400> 22 Gly Gly Thr 1 Gly Thr Ala Cys Gly Cys Thr Cys Ala Gly Ala Ala Gly Ala Cys Gly 5 10 <210> 23 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 23 Ala Ala Gly Cys Ala Gly Thr Gly Gly Thr Ala Ala Cys Ala Ala Cys 1 5 10 Gly Cys Ala Gly Ala Gly Thr <210> 24 WO 01/30817 PCT/AU00/01227 -46- <211> <212> <213> <220> <223> 22

PRT

Artificial Sequence Description of Artificial Sequence:primer <400> 24 Cys Gly Gly Gly Ala Thr Ala Thr Cys Ala Cys Thr Cys Ala Gly Cys 1 5 10 Ala Thr Ala Ala Thr Gly <210> <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> Cys Gly Gly 1 Thr Cys Gly Gly Ala Thr Cys Cys Ala 5 Thr Gly Gly Cys Gly Gly Cys 10 Thr Cys Ala Gly Cys Ala <210> 26 <211> 29 <212> PR' <213> Ar <220> <223> De

T

tificial Sequence scription of Artificial Sequence:primer WO 01/30817 WO 0130817PCT/AUOO/01 227 -47 <400> 26 Cys Gly Cys Thr Cys Cly Ala Gly Thr Thr Ala Ala Ala Ala Gly Thr 1 5 10 Thr Cys Gly Ala Gly Cys Thr Cys Thr Cys Thr Thr Gly <210> 27 <211> 3111 <212> DNA <213> Blomija tropicalis <400> 27 gacatattcg gaacaacaag cccaagtggc gtagacgatc aaatttacaa tgatttgaaa cagtcgtgct cacccgactt t cgacaacgt acgtttggag ctcggaattg tgctcaccat tcaggtgcaa tttcgaattg ggtagaaaag aactgttgtt caaggaagtt tgcacaacaa cttggagaac agaggaatct tgcttcatgg acttcgacga gaacaaatgt aatcaaactc gtgtcccaat gtacactgca aacgaaatca gtgtacacca caataatcaa ggacacggcg gaggacaaga gt tgaacgag gaggccggag gctaagcttc ttgagacaaa aaggcaaaga ttggctcagg ttggaataca gaggttaccg catgaataca ttggaggaca cacgcccaca gaggcccgat aaatccaaat aagatggccc agtgcattgg tcgtctagtg acatacacac ttcgcaagtc gtctcatcct ttttccaata cgacaatggc gcgacatttc ttcgactttt aaaagtcgga gttcgagcga gaaagttgct agcaccaagc acaagtcgga ttgagactgc ccgtacatga ctcaccgaca agatttcatt cccgacaccg cat tggaagt tagaattgga acgaagccga agaagatttc aaaagcaaaa agttgtgcac atacacactc tctcacatca acttctaaag agacataagg ggctcgatca catcgaatat gtccgaagat tattaccgtt aagcgttacc cgaagatgtc tgccattcaa taaggagaag caacaaggac gttgaacatt acgattgtcg ggacaatgct attggaggat tgaattggaa acgacaattg acttcaagct ggaatatgag ggctcgactc tcgtcgtcga accacacata tct tc accag cattgaacac gaatagaacc gcaaagtata ggaac cga tt ttggaaagcg caattgatga gagatgaaca catatggaaa gagatgcaag caaaagttcc aagttggtcg a agat tgaag caagagaatt aaccacttga gaagaacgaa tcattgaagg actaaagcta cacgttgatg gaacaattgg caatcggagg gtattcgacc ttcgacctgt aagcagagta ctttctacac aatattgatt tgtatcagtc tgggcgcact aacgggagct act tgaccga agaaacgaga gcgaggagac atcaattgga aagccgaagt ctcagaagac aaatcaatcg cggaattgat agggacaaat aacgatcaag tacaattgga atggcgatgc aggttgaaga aagcattgtt cagaagtttt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 WO 01/30817 WO 0130817PCT/AUOO/01227 48 gatcatggac attggagaag acaaactcaa cgaaaagttg tttggctgaa cgagat caaa aaccttgcga acgacacgac acaataccaa gaagaccgag gtcgttggat tctccaaatc tgttgatcaa gatgcgagtc agaggcagt t gagcaaattg actccgaatc cttgctcgaa aaccgaagta tggtaaacga agaagaacga caaggaattg gtccgataaa aatgagccaa agaccgagct ggtcaccacc ctcgaacttt tttgaaattc caacctctta ttggaaaagg atcaacctcg aaggatctc cgagaccaaa gctaaatctc cgat tggaga aaacaagaag tatgagaaac at tgaaattg gttgcacgac gccgctaata actgagcttc ttgggtgtca aac tt ggaac gtccgagtca gagactgaat tctgacgaac tcagaaacgg cgaaatctcc gtcatcgcca cgacgacacg ttgcttcaaa ttgaacgaaa caaaacttga gatcaagctg agtcaagttc taaataacag aaaccaaaca aacacacctc ctaccgcaca acctcaagag gtgtcaagat aggaagcatt agt tgaatga acgagcgaga aggcaaagaa gattggcaca aacaattgaa tcaagaagaa aggctaacat aagcccatta ctcaacgacg aagcacttcg acgaacttac tctctgctct gagtgcaaaa aacgtgtcac aaatccgaat agttggaatc cagaaactga acgaggagga aggtcaaggt cccgtgtccg aatccaattt ctggcggtac catgcaacaa t agat ataaa gattagcgtt tgcccaggca caagttggag tgcagac ttg ggcccgtgag cgcccaccga agaattggct tcaacgattg aaaggaagag catgcgattg gtaccaagca cgacttgcag tgatgaggta atgccaagca tgctaaacga aaccatcaat tcaaaacgat acttaccatt aaaattggaa tgaagaagtt gcgaatccgt aaagatgctt ccataaacaa ttacaaacga acgattccaa gtcgttcatc tcgacaagtc attcattatt tctaataaaa taccaccact ttggagaaac gaggtcagca cagaaattgc aacaagaaac cgaatccatg gccgcataca actgccgagt gagattgaag gctgaggctg caaatcactg aagacgatca catcgtcaat ttgaccgccg gctgccgaac gtcaacctcg tacgatgaag gaggtcaagt acaatcaaga gaggctaatg gatgttgaga cgaaagaagg attcaattgt caaatgcaag cgagagttgg cgagccaaac tttgtcacgg tcatataaat aaaaagtttt atcaccacct gcgtttccca tgttgttgga aacacgagta tcgctgatga aacaagagat aggaagctga tggcacagac ctcttcgaaa aagctaaatt a at tggaa tt agaagcaagc tgcaacaagc aattggagga aaatgcacga c C Ccggccaa tacacaagga cgacaaagga aatcat tgga cattggctgg t tgaggtiga atcatcgtgt tgcaagaaat aacaggaggg aagccgccga atcgatcgtg aacaagagag cgaacaataa aaaaaaatca c 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3111 <210> 28 <211> 1014 <212> DNA <213> Blomia tropicalis <220> <221> CDS WO 01/30817 WO 0130817PCT/AUOO/01227 -49 <222> <400> 28 ataaaa aig gag gcc Met Giu Ala 1 aag gat aat gec atc Lys Asp Asn Ala Ilie atc aag aag aag atg cag gca.

Ile Lys Lys Lys Met Gin Ala atg aag ctc gag Met Lys Leu Glu ega. get gag att Arg Ala Giu Ile gaa. caa aag tcg Glu Gin Lys Ser gat gcc aac cte Asp Ala Asn Leu gcc gag aag tee gaa gag gaa gte cgt Ala Glu Lys Ser Glu Glu Glu Val Arg gee ctt Ala Leu caa aag aag att caa caa att gaa aat gaa ttg gat eaa Gin Lys Lys Ile Gin Gin Ile Giu Asn Glu Leu Asp Gin gte eaa gaa.

Val Gin Glu aag tca etc Lys Ser Leu teg ttg ace Ser Leu Thr eaa gee aat acc aaa ttg gaa gag aag Gin Ala Asn Thr Lys Leu Giu Giu Lys eaa aeg Gin Thr gee gaa ggt gat gte get get ett aae ega cgt ate caa. etc Ala Giu Gly Asp Val Ala Ala Leu Asn Arg Arg Ile Gin Leu gaa gag gat ttg gaa ega tee gaa gag Glu Glu Asp Leu Giu Arg Ser Glu Glu 100 etc aag gtt gee Leu Lys Val Ala gee aag etc gaa gag get tea. cac tet gee gat gaa. tet gaa.

Ala Lys Leu Giu Giu Ala Ser His Ser Ala Asp Glu Ser Giu 115 120 cgt aag atg ett gaa. eat ega age ate ace gat gag gaa egt Arg Lys Met Leu Glu His Arg Ser Ile Thr Asp Glu Glu Arg egt atg Arg Met 125 atg gat Met Asp 130 135 WO 01/30817 WO 0130817PCT/AUOO/01227 rsr71 rct- Gly Leu Ser Gin Leu Lys ac cat atq ato qct qaa qat qct Ala Arg Met Met Ala Glu Asp Ala 155 gac cga Asp Arg 160 aaa tat gat gaa Lys Tyr Asp Glu gcc cgt aaa ttg Ala Arg Lys Leu atg gtt gag gct Met Val Glu Ala ctt gaa cgt gct Leu Giu Arg Ala gag cga gcc gaa Glu Arg Ala Glu ggt gaa aca aag Gly Glu Thr Lys gtt gaa ctc gaa Val Glu Leu Giu gag ttg cga gtc Giu Leu Arg Val ggt aac aac ttg Gly Asn Asn Leu aaa tcg Lys Ser 205 cic gaa gtc Leu Glu Val caa caa atc Gin Gln Ile 225 gaa gag aag gcc Giu Glu Lys Ala caa cgt gag gaa Gin Arg Glu Glu gct tat gag Ala Tyr Giu 220 gag gec cga Glu Ala Arg cga atg atg acc Arg Met Met Thr aag etc aag gag Lys Leu Lys Giu gct gaa Ala Giu 240 ttt gca gaa cga Phe Ala Glu Arg gtt caa aag ttg Val Gin Lys Leu aag gaa gtc gat Lys Glu Val Asp ctg gaa gac gag Leu Giu Asp Glu gtg cac gag aag Val His Giu Lys aaa tac aaa tea Lys Tyr Lys Ser teg gac gaa ttg Ser Asp Glu Leu eag aca ttt gc Gin Thr Phe Ala gaa Glu 280 etc act ggt tat Leu Thr Gly Tyr taagcttata tacgceatcg atcgaegeeg accaacagc caaaaacaac caagttgtca 918 WO 01/30817 WO 0130817PCT/AUOO/01227 -51 caaataatat aatgatggag acgaatgcaa atcaaa 2.0124 <210> 29 <211> 284 <212> PRT <213> Biomia tropicalis <400> 29 Met Glu 1 Asn Ala Ala Ile Lys 5 Ile Asp Arg Lys Lys Met Gin Met Lys Leu Giu Lys Asp Ala Glu Ile Oin Lys Ser Asn Leu Arg Lys Ile Gin Ala Gin Giu Lys Ser Ile GlU Asn Thr Lvs Leu Glu Val Arg Leu Gin Lye Giu Ser Leu Leu Asp Gin Thr Gin Ala Asn Giu Giu Lys Ser Leu Gin Ala Glu Giy Asp Ala Leu Asn Ile Gin Leu Ile Giu Giu Asp Leu Leu Glu Giu 115 Met Leu Giu 130 Ser Giu Giu Lys Val Ala Ala Ser His Scr His Arg Ser Ile 135 Giu Ser Giu Thr Ala Lys 110 Met Arg Lys Asp Gly Leu Asp Giu Giu WO 01/30817 WO 0130817PCT/AUOO/01227 52 Ser Gin Leu Lys Ala Arq Met Met Glu Asp Ala Asp Lys Tyr Asp Glu Ala Arg Lys Leu Met Val Glu Ala Asp Leu 175 Glu Arg Ala Glu Glu Arg Ala Glu 180 Gly Glu Thr Lys Ile Val Giu 190 Ser Leu Glu Leu Glu Glu 195 Giu Leu Arg Val Gly Asn Asn Leu Val Ser Glu Giu Lys Ala 210 Gin 215 Gin Arg Giu Glu Tyr Giu Gin Gin Arg Met Met Thr Lys Leu Lys Glu Ala Giu Ala Arg Ala 235 Giu 240 Arg Leu 255 Phe Ala Giu Arg Val Gin Lys Leu Gin Lys Giu Val Asp 250 Giu Asp Glu Leu Val His Giu Lys 260 Lys Tyr Lys Ser Ile Ser Asp 270 Giu Leu Asp 275 Gin Thr Phe Ala Leu. Thr Gly Tyr <210> <211> <212> <213> <220> <221> <222> 852

DNA

Blomia tropicalis

CDS

WO 01/30817 WO 0130817PCT/AUOO/01227 53 <400> atg gag Met Glu 1 gcc atc aag Ala Ile Lys 5 aag aag atg cag L~ys Lys Met Gin atg aag ctc gag Met Lys Leu Giu aag gat Lys Asp aat gcc atc Asn Ala Ile cga gct gag att Arg Ala Glu Ile gaa caa aag tcg Giu Gin Lys Ser cga gat gcc Arg Asp Ala ctt caa aag Leu Gin Lys aac ctc aga Asn Leu Arg gcc gag aag tcc gaa gag gaa gtc cgt Ala Giu Lys Ser Giu Giu Giu Val Arg aag att Lys Ile caa caa att gaa aat gaa ttg gat caa Gin Gin Ilie Giu Asn Glu Leu Asp Gin caa gaa tcg ttg Gin Giu Ser Leu acc caa gcc aat acc aaa ttg gaa gag aag Thr Gin Ala Asn Thr Lys Leu Glu Giu Lys gcc gaa ggt gat gtc gct gct ctt aac cga Ala Giu Gly Asp Vai Ala Ala Leu Asn Arg 90 aag tca ctc caa Lys Ser Leu Gin cgt atc caa ctc Arg Ile Gin Leu att gaa Ile Glu gag gat ttg gaa cga tcc gaa gag Glu Asp Leu Glu Arg Ser Glu Glu 100 ctc aag gtt gcc Leu Lys Val Ala acc gcc aag Thr Ala Lys 110 atg cgt aag Met Arg Lys ctc gaa gag Leu Glu Giu 115 gct tea cac tct gcc gat gaa tct gaa Ala Ser His Ser Ala Asp Giu Ser Giu 120 atg ctt gaa cat cga agc Met Leu Giu His Arg Ser 130 ace gat gag gaa cgt atg gat ggc ctc Thr Asp Giu Glu Arg Met Asp Gly Leu 432 WO 01/30817 WO 0130817PCT/AUOO/01227 -54gag agt caa tta aaa Glu Ser Gin Leu Lys 145 gee cgt atg atg Ala Arg Met Met gaa gat get gac Glu Asp Ala Asp aaa tat gat gaa Lys Tyr Asp Glu gcc cgt aaa ttg Ala Arg Lys Leu atg gtt gag get Met Val Glu Ala gat ctt Asp Leu 175 gaa egt get gaa gag ega gcc gaa acc ggt gaa aca aag Glu Arg Ala Glu Glu Arg Ala Glu Thr Gly Glu Thr Lys att gtt gaa Ile Val Glu 190 tcg ctc gaa Ser Leu Glu 576 ctc gaa gaa Leu Glu Glu 195 gag ttg cga gte Glu Leu Arg Val ggt aac aae ttg Gly Asn Asn Leu 624 gtc agc Val Ser 210 gaa gag aag gec caa eaa egt gag gaa Glu Glu Lys Ala Gin Gin Arg Glu Glu 215 tat gag caa caa Tyr Glu Gin Gin cga atg atg ace Arg Met Met Thr aag etc aag gag Lys Leu Lys Giu gag gcc ega get Glu Ala Arg Ala ttt gca gaa cga Phe Ala Glu Arg tcg Ser 245 gtt caa aag ttg Val Gin Lys Leu aag gaa gte gat cga etg Lys Glu Val Asp Arg Leu 255 gaa gac gag Glu Asp Glu gtg cac gag aag Val His Giu Lys aaa tac aaa tea Lys Tyr Lys Ser ate teg gae Ile Ser Asp 270 gaa ttg gac Giu Leu Asp 275 eag aca ttt gee Gin Thr Phe Ala ete act ggt tat 852 Leu Thr Gly Tyr <210> 31 WO 01/30817 WO 01/08 17PCT/AUOO/01227 55 <211> 284 <212> PRT <213> Biomia tropicalis <400> 31 Met 1 Glu Ala Ile Lys Lys Met Gin Asn Ala Ile Asn Leu Arg Lys Ile Gin Ala Giu Ile Ala Met Lys 10 Glu Gin Lys Giu Val Arg Leu Giu Ser Lys Asp Glu Lys Ser Arg Asp Ala Leu Gin Lye Glu Ser Leu Gin Ile Giu Leu Asp Gin Thr Gin Ala Asn Thr Giu Giu Lys Ser Leu Gin Ala Giu Gly Asp Ala Leu Asn Arg Ile Gin Leu Ile Giu Giu Asp Leu Leu Giu Giu 115 Met Leu Giu 130 Giu Ser Gin Ser Glu Giu Lys Vai Ala Ser His Ser Giu Ser Glu Thr Ala Lys 110 Met Arg Lys Asp Giy Leu His Arg Ser Leu Lye Giu 150 Glr Vsp Aar yLeVaGiAl Asp Glu Glu Arg Met Met Asp Ala Asp Tyr Asp Arg Lys Leu Val Glu Ala WO 01/30817 PCT/AU00/01227 -56- Glu Arg Ala Leu Glu Glu 195 Glu Arg Ala Glu Thr Gly 185 Glu Thr Lys Ile Val Glu 190 Ser Leu Glu Glu Leu Arg Val Gly Asn Asn Leu Val Ser 210 Glu Glu Lys Ala Gin Arg Glu Glu Tyr Glu Gin Gin Arg Met Met Thr Lys Leu Lys Glu Glu Ala Arg Ala Phe Ala Glu Arg Val Gin Lys Leu Lys Glu Val Asp Arg Leu 255 Glu Asp Glu Val His Glu Lys Lys Tyr Lys Ser Ile Ser Asp 270 Glu Leu Asp 275 Gin Thr Phe Ala Leu Thr Gly Tyr <210> 32 <211> 284 <212> PRT <213> Dermatophagoides farinae <400> 32 Met Glu Ala Ile Lys Lys Lys Met Gin Ala Met Lys Leu Glu Lys Asp Asn Ala Ile Asp Arg Ala Glu Ile Asn Leu Arg Ala Glu Lys Ser Glu Glu Gin Lye Ala Arg Asp Ala Glu Glu Val Arg Ala Leu Gin Lys WO 01/30817 WO 0130817PCT/AUGO/0 1227 57 Lys Ile Ser Ala Gin Gin Ilie Giu Glu Leu Asp Gin Ala Asn Thr Glu Glu Lys Val Lys Ile Gin Glu Gin Leu Aia Leu Gin Thr Ala Giu Gly Asp Ala Leu Asn Gin Giu Asp Leu Leu Giu Glu 115 Met Leu Giu Arg Ser Giu Glu Lys Ile Ala Leu Ile Giu Thr Ala Lys 110 Met Arg Lys Asp Gly Leu Ser Gin Ser Glu Ser Giu His Arg Ser Asp Giu Glu 130 Glu Asn Gin Leu Lye Arg Met Met Asp Ala Asp Tyr Asp Glu Arg Lys Leu Val Giu Ala Asp Leu 175 Glu Arg Ala Leu Glu Giu 195 Val Ser Glu Arg Ala Giu Glu Ser Lys Leu Arg Val Asn Asn Leu Ile Val Glu 190 Ser Leu Glu Glu Gin Gin Glu Lys Aia 210 Ile Arg 225 Gin Arg Glu Glu Leu Lye Glu Ala 235 Ile Met Thr Ala Arg Ala Phe Ala Glu Arg Ser Val Gin Lys Leu Gin Lys Glu Val Asp Arg Leu WO 01/30817 PCT/AU00/01227 -58- Glu Asp Glu Leu Val His Glu Lys Glu 260 265 Glu Leu Asp Gin Thr Phe Ala Glu Leu 275 280 Lys Tyr Lys Ser Ile Ser Asp 270 Thr Gly Tyr <210> 33 <211> 284 <212> PRT <213> Lepidoglyphus destructor <400> 33 Met Glu Ala 1 Asn Ala Ile Asn Leu Arg Ile Lys 5 Asn Lys Met Gin Met Lys Leu Glu Lys Asp Arg Ala Glu lie Glu Gin Lys Ser Arg Asp Ala Leu Gin Lys Ala Glu Lys Ser Glu Glu Val Arg Lys Ile Gin Gin Ile Glu Asn Glu Leu Asp Gin Gin Glu Ser Leu Gin Ala Asn Thr Leu Glu Glu Lys Glu Lys Ser Leu Gin Thr Ile Glu Ala Glu Gly Asp Ala Ala Leu Asn Arg Arg Ile Gin Leu Glu Asp Leu Arg Ser Glu Gly Leu Lys Ile Ala Thr Ser Lys 110 Leu Glu Glu 115 Ala Ser Gin Ser Asp Glu Ser Glu Arg Met Arg Lys WO 01/30817 WOO1/0817PCT/AUOO/01227 -59- Met Leu 130 Glu His Arg Ser Thr Asp Glu Glu Met Glu Gly Leu Ser Gin Leu Lys Ala Arg Met Met Glu Asp Ala Asp Arg 160 Lys Tyr Asp Giu Ala Arg Lys Leu Met Val Glu Ala Asp Leu 175 Glu Arg Ala Giu Giu Arg Ala Giu 180 Gly Giu Ser Lys Ile Val Giu 190 Leu Giu Glu 195 Glu Leu Arg Val Gly Asn Asn Leu Ser Leu Glu Val Ser 210 Giu GiU Lys Ala Gin Gin Arg Glu Giu Ala Tyr Glu Gin Gin 215 220 Lys Leu Lys Glu Ala Giu Ala Arg Ala Giu 235 240 Arg Ile Met Thr Phe Ala Giu Arg Ser Val Gin Lys Leu 245 Lys Glu Val Asp Arg Leu 255 Glu Asp Glu Val His Giu Lys Glu Lys Tyr Lys Ser 265 Ile Ser Asp 270 Giu Leu Asp 275 Gin Thr Phe Ala Leu Thr Gly Tyr <210> 34 <211> 284 <212> PRT <213> Dermatophagoides pteronyssinus <400> 34 Met Giu Ala Ile Lys Lys Lys Met Gin Ala Met Lys Leu Glu Lys Asp WO 01/30817 WO 0130817PCT/AUOO/01227 60 Ala Ile Asp Ala Giu Ile Ala Gin Lys Ala Arg Asp Ala Leu Gin Lys Glu Gin Leu Asn Leu Arg Lys Ile Gin Ala Giu Lys Ser Giu Glu Giu Vai Arg Gin Ile Giu Giu Leu Asp Gin Ser Ala Ala Asn Thr Giu Giu Lys Ala Leu Gin Aia Giu Gly Asp Val Ala LeU Asn Arg Ile Gin Leu Ile Giu Giu Asp Leu Leu Giu Giu 115 Met Leu Giu 130 Giu Asn Gin 2145 Lys Tyr Asp Glu Arg Ala Arg Ser Glu Giu Lys Ile Ala Ser Gin Ser Glu Ser Glu Thr Ala Lys 110 Met Arg Lys Giu Gly Leu His Arg Ser Asp Giu Giu Leu Lys Giu Val 165 Giu Giu 180 Arg Met Met Asp Ala Asp Arg Lys Leu Val Glu Ala Asp Leu 175 Val Giu Arg Ala Glu Giu Ser Lys Leu Giu Giu Giu Leu Arg Val Vai Gly Asn Asn Leu Lys ser Leu Giu WO 01/30817 WO 0130817PCT/AUOO/0 1227 -61- Val Ser 210 Giu Glu Lys Ala Gin Arg Giu Giu His Giu Gin Gin Ile 225 Arg Ile Met Thr Lys Leu Lys Glu Ala Giu Ala Arg Ala 235 Phe Aia Giu Arg Val Gin Lys Leu Lys Giu Val Asp Arg Leu 255 Giu Asp Giu Val His Giu Lys Lys Tyr Lys Ser Ile Ser Asp 270 Giu Leu Asp 275 Gin Thr Phe Ala Leu Thr Gly Tyr <210> <211> 284 <212> PRT <213> Dermatophagoides pteronyssinus <400> Met Giu Ala Ile Lys Asn Lys Met Gin 1 5 Met Lys Leu Giu Lys Asp Asn Ala Ile Asn Leu Arg Arg Ala Giu Ile Glu Gin Lys Ala Arg Asp Ala Leu Gin Lys Ala Glu Lys Ser Giu Giu Val Arg Lys Ile Gin Gin Ile Glu Giu Leu Asp Gin Gin Giu Gin Leu Se r Ala Ala Asn Thr Leu Giu Giu Lys Lys Ala Leu Gin Ala Giu Gly Asp Val Ala Ala Leu Asn Arg Axg Ile Gin Leu Ile GiU WO 01/30817 WO 0130817PCT/AUOO/01227 62 Giu Asp Leu Leu Glu Glu 115 Met Leu Glu Ser Glu Glu Lys Ile Ala Thr Ala Lys 110 Met Arg Lys Glu Gly Leu Ser Gin Ser Glu Ser Glu His Arg Ser Asp Giu Glu 130 Glu Ann Gin Leu Lys Arg Met Met Asp Ala Asp Tyr Asp Giu Arg Lys Leu Val GiU Ala Asp Leu 175 Glu Arg Ala Leu Giu Glu 195 Val Ser Glu Arg Ala Giu Glu Ser Lys Leu Arg Val Asn Aen Leu Ile Val Giu 190 Ser Leu Giu Glu Gin Gin Glu Lys Ala Gin 215 Lys Arg Giu Glu Ile Met Thr Leu Lys Glu Ala Arg Ala Ala Giu Arg Se r 245 Val1 Gin Lys Leu Giu Val Gly Arg Leu 255 Ser Asp Giu Asp Glii Glu Leu Asp 275 His Glu Lys Tyr Lys Ser Thr Phe Ala Thr Giy Tyr WO 01/30817 WO 0130817PCT/AUDO/01 227 63 <210> 36 <211> 408 4212> DNA <213> Blomia tropicalis <400> 36 acaatgaagt cacaagccaa aaccatgcta ttgaatgaaa tgcgccatga aacattttgg gat ttgaagg tcgccatcgt agaaggatga tcgaaaaggg acaagagcaa tcgaaggagc aacgattcaa aaaccgaaca tcttattgcc tttccgaaac agaacatcaa ggaattgcaa ccaaggagct ctacgaagag aaaagtgaag tgctttgccg gaattcgatc ttgctttact gagaaaatca ttggaacgtg gctcaaactc gatattcaaa cttcggtttt acttgttgat tgcaacacca ttcgagaact aattgaagcg tcagcaagat cccaataa ggctcaagag cgaacaggca actcgacgaa tgatgttgtt aactgatctt cttgcttaag <210> 37 <211> 117 <212> PRT <213> Biomia tropicalis <400> 37 Gin Giu His Lys Pro Lys Lys Asp Asp 1 5 Arg Asn Glu Phe Asp His is Leu Leu Ile Gin Ala Asn His Ile Glu Lys Gly Glu His Gin Leu Leu Tyr Leu Gin His Gin Asp Giu Leu Asn Aen Lys Ser Lys Giu Leu Gin Giu Lys Ile Ile Arg Glu Leu Asp Val Val Cys Ala 55 Gly Ala Leu Glu Arg Giu Leu Lye Arg Thr Ile Giu Gly Ala Asp Leu Asi Ilie Leu Giu Arg Phe Aen Tyr Giu Giu Ala Gin Thr Leu WO 01/30817 WO 0130817PCT/AUGO/01227 64 Ser Lys Ile Leu Leu Lys Asp Leu Lys Giu Thr Glu Gin Lys Val Lys 100 105 110 Asp Ile Gin Thr Gin 115 <210> 38 <211> 464 <212> DNA <213> Blomia tropicalis <400> 38 acaatgaagt cacaagccaa aacagttttt atgctatcga atgaaaacaa ccatgatcga ttttggaacg tgaaggaaac tcgccatcgt agaaggatga tttttgaaag aaagggagaa gagcaaggaa aggagcc caa attcaactac cgaacaaaaa tcttattgcc tttccgaaac gttaataaaa catcaattgc t tgcaagaga ggagctttgg gaagaggctc gtgaaggata tgctttgccg gaattcgatc aagtaatcaa tttacttgca aaatcattcg aacgtgaatt aaactctcag ttcaaaccca ct tcggt t tt acttgttgat agaaactttt acaccaactc agaacttgat gaagcgaact caagatcttg at aa ggctcaagag cga acagg ta taggcaaacc gacgaattga gttgtt tgcg gatcttaaca cttaaggatt <210> 39 <211> 462 <212> DNA <213; Blomia tropicalis <400> 39 acaatgaagt cacaagccaa aacagttttt gctatcgaaa gaaaacaaga atgatcgaag ttggaacgat tcgccatcgt agaaggatga tttgaaaggt aggg tgaaca gcaaggaatt gagcccaagg tcaactacga tcttattgcc tttccgaaac taataaaaaa tcaattgctt gcaagagaaa agctt tggaa agaggctcaa tgctttgccg gaattcgatc gtaatcaaag tacttgcaac at cat tcgag cgtgaattga actctcagca cttcggtttt acttgttgat aaacttttta accaactcga aacttgatgt agcgaactga agatcttgct ggctcaagag cgaacaggta ggcaaaccat cgaattgaat tgtttgcgcc tcttaacatt taaggatttg WO 01/30817 WOO1/0817PCT/AUGO/01227 aaggaaaccg aacaaaaagt gaaggatatt caaacccaat aa <210> <211> 462 <212> DNA <213> Blorija tropicalis <400> acaatgaagt cacaagccaa aacagttttt gctatcgaaa gaaaacaaga atgatcgaag ttggaacgat aaggaaaccg tcgccatcgt agaaggatga tttgaaaggt agggtgaaca gcaaggaatt gagcccaagg tcaactacga aacaaaaagt tcttattgcc tttccgaaac taataaaaaa tcaattgatt gcaagagaaa agctttggaa agaggctcaa gaaggatatt tgctttgccg gaattcgatc gtaataaaag tacttgcaac atcattcgag cgtgaattga actctcagca caaacccaat cttcggtttt acttgttgat aaacttttta accaactcga aacttgatgt agcgaactga agatcttgct aa ggctcaagag cgaacaggta ggcaaaccat cgaattgaat tgtttgcgcc tcttaacatt taaggatttg <210> 41 <211> 462 <212> DNA <213> Blomia tropicalis <400> 41 acaatgaagt c acaagccaa aacagttttt gctatcgaaa gaaaacaaga atgatcgaag ttggaacgat aaggaaaccg tcgccatcgt agaaggatga tt tgaaaggt agggagaaca gcaaggaatt -gagcccaagg tcaactacga aacaaaaagt tcttattgcc tttccgaaac taataaaaaa tcaattgctt gcaagagaaa agc tt tggaa agaggc tcaa gaaggatatt tgctttgccg gaattcgatc gtaatcaaag tacttgcaac atcattcgag cgtgaat tga actctcagca caaacccaat cttcggtttt actggttgat aaacttttta accaactcga aacttgatgt agcgaac tga agatcttgct aa ggctcaagag cgaacaggta ggcaaaccat cgaattgaat cgt ttgcgcc tcttaacatt taaggatttg <210> <211> <212> WO 01/30817 WO 0130817PCT/AUOO/01227 -66- <213> Blomia tropicalis <400> 42 acaatgaagt cacgagccaa aacagttttt gctatcgaaa gaaaacaaga atgatcgaag ttggaacgat aaggaaaccg tcgccatcgt agaagga tga tttgaaaggt agggtgaaca gcaaggaatt gagcccaagg tcaactacga aacaaaaagt tcttattgcc tttccgaaac taataaaaaa tcaattgatt gcaagagaaa agctttggaa agaggctcaa gaaggatatt tgctttgccg cttcggtttt gaattcgatc act tgttgat gtaatcaaag aaacttttta tacttgcaac accaactcga atcattcgag aacttgatgt cgtgaattga agcgaactga actctcagca agatcttgct caaacccaat aa ggctcaagag cgaacaggta ggcaaaccat cgaattgaat tgtttgcgcc tcttaacatt taaggatttg <210> 43 <211> 462 <212> DNA <213> Blomia tropicalis <400> 43 acaatgaagt cacaagccaa aacagttttt gctatcgaaa gaaaacaaga atgatcgaag ttggaacgat aaggaaaccg tcgccatcgt agaaggatga tttgaaaggt agggagaaca gcaaggaatt gagcccaagg tcaactacga aacaaaaagt tcttattgcc tttccgagac taataaaaaa tcaattgctt gcaagagaaa agctttggaa agaggctcaa gaaggatatt tgctttgccg gaattcgatc gtaatcaaag tacttgcaac atcattcgag cgtgaattga actctcagca caaaccaaat cttcggt t tt acttgttgat aaact t ttta accaactcga aacttgatgt agcgaactga agatcttgct aa ggctcaagag cgaacaggta ggcaaaccat cgaattgaat tgtttgcgcc tcttaacatt taaggatt tg <210> 44 <211> 462 <212> DNA <213> Blomia tropicalis <400> 44 acaatgaagt tcgccatcgt tcttattgcc tgctttgccg cttcggtttt ggctcaagag cacaagccaa agaaggatga tttccgaaac gaattcgatc acttgttgat cgaacaggta 120 aacagttttt tttgaaaggt taataaaaaa gtaatcaaag aaacttttta ggcaaaccat 180 WO 01/30817 WO 0130817PCT/AUOO/01227 67 gctatcgaaa agggagaaca tcaattgctt qaaaacaaqa gcaaggaatt gcaagagaaa atgatcgaag gagcccaagg agctttggaa ttggaacgat tcaactacga agaggctcaa aaggaaaccg aacaaaaagt gaaggatatt tacttgcaac accaactcga cgaattgaat 240 atcattcgag aacttgatgt tgtttgcgcc 300 cgtgaattga agcgaactga tcttaacatt 360 actctcagca agatcttgct taaggatttg 420 caaacccaat aa 462 <210> <211> 462 <212> DNA <213> Blomia tropicaliS <400> acaatgaagt cacaagc caa aacagttttt gctatcgaaa gaaaacaaga atgatcgaag ttggaacgat aaggaaaccg tcgccatcgt agaaggatga tttgaaaggt agggtgaaca gcaaggaatt gagcccaagg tcaactacga aacaaaaagt tcttattgcc tttccgaaac taataaaaaa tcaattgctt gcaagagaaa agc tttggaa agaggctcaa gaaggatatt tgctttgccg gaattcgatc gtaatcaaag tacttgcaac atcattcgag cgtgaattga actctcagca caaacccaat cttcggtttt acttgttgat aaacttttta accaactcga aacttgatgt agcgaactga agatcttgct aa ggctcaagag cgaacaggta ggcaaaccat cgaattgaat tgtttgcgcc tcttaacatt taaggatttg <210> 46 <211> 462 <212> DNA <213> Blomia tropicalis <400> 46 acaatgaagt cacaagccaa aacagttttt gctatcgaaa gaaaacaaga atgatcgaag ttggaacgat aaggaaaccg tcgccatcgt agaaggatga tttgaaaggt agggagaaca gcaaggaatt gagcccaagg tcaactacga aacaaaaagt tcttattgcc tttccgaaac taataaaaaa tcaattgctt gcaagagaaa agctttggaa agaggctcaa gaaggatatt tgctttgccg gaattcgatc gtaatcaaag cacttgcaac atcattcgag cgtgaattga actctcagca caaacccaat cttcggtttt acttgttgat aaacttttta accaactcga aacttgatgt agcgaactga agatcttgct aa ggctcaagag cga acaggta ggcaaaccat cgaattgaat tgtttgcgcc tcttaacatt taaggatttg WO 01/30817 WO 0130817PCT/AUO/01227 68 <210> 47 <211> 463 <212> DNA <213> Blomia tropicalis <400> 47 acaatgaagt cacaagccaa aacagttttt tgctatcgaa tgaaaacaag catgatcgaa tttggaacga gaaggaaacc tcgccatcgt agaaggatga ttttgaaagg aagggagaa c agcaaggaat ggagcccaag ttcaactacg gaacaaaaag tcttattgcc tttccgaaac ttaataaaaa atcaattgct tgcaagagaa gagctttgga aagaggctca tgaaggatat tgctttgccg gaattcgatc agt aat caa a ttacttgcaa aatcattcga acgtgaattg aac tc tcagc tcaaacccaa cttcggtttt acttgttgat gaaacttttt caccaactcg gaacttgatg aagcgaactg aagatcttgc taa ggc tcaagag cgaacaggta aggcaaacca acgaattgaa ttgtttgcgc atc tta acat ttaaggattt <210> 48 <211> 408 <212> DNA <213> Blomia tropicalis <400> 48 caacagcaca caggcaaaca aaccatgcta t tga atgaaa tgcgccatga aacattttgg gatttgaagg agccaaagaa gttttttttg tcgaaaaggg acaagagcaa tcgaaggagc aacqattcaa aaaccgaaca ggatgatttc aaaggttaat tgaacatcaa ggaattgcaa ccaaggagct ctacgaagag aaaagtgaag cgaaacgaat aaaaaagtaa ttgctttact gagaaaatca ttggaacgtg gctcaaactc gatattcaaa tcgatcactt tcaaagaaac tgcaacacca ttcgagaact aattgaagcg tcagcaagat cccaataa gttgatcgaa tttttaggca a ctcgacgaa tgatgttgtt aactgatctt cttgcttaag <210> <211> <212> <2 13> 49 117

PRT

Blomia tropicalis <400> 49 WO 01/30817 PCT/AU00/01227 -69- Gln Glu His Lys 1 Lys Lys Asp Asp Phe Arg Asn Glu Phe Asp His Leu Leu Ile Glu Gin Ala Asn His Ile Glu Lys Gly Glu His Gin Leu Leu Tyr Leu Gln His Gin Leu Asp Glu Leu Asn Glu Asn Lys Ser Lys Glu Leu Gin Glu Lys Ile Ile Arg Glu Leu Asp Val Val Cys Ala Met Ile Glu Gly Ala Gin Gly Ala Leu Glu Arg Glu Leu Lys Arg Asp Leu Asn Ile Glu Arg Phe Asn Tyr Glu Glu Ala Gin Thr Leu Ser Lys Ile Leu Leu Lys Asp Leu Lys Glu Thr Glu Gin Lys Val Lys 110 100 105 Asp Ile Gin Thr Gin 115 <210> <211> 117 <212> PRT <213> Blomia tropicalis <400> Gin Glu His Lys Pro Lys Lys Asp Asp Arg Asn Glu Phe Asp His Trp Leu Ile Glu Gin Ala Asn His Ala Ile Glu Lys Gly Glu His Gin Leu Leu Tyr Leu Gin His Gin Leu Asp Glu Leu Asn Glu Asn Lys Ser WO 01/30817 WO 0130817PCT/AUOO/01227 Lys Giu Met Ile Gin Giu Lys Ile Arg Glu Leu Asp Val Ala Leu Glu Arg Giu Leu Giu Gly Ala Val Cys Ala Lys Arg Thr Gin Thr Leu Lys Val Lys 110 Leu Asn Ile Arg Phe Asn Tyr Giu Giu Ala Giu Thr Giu Gin Ser Lys Ilie Asp Ile Gin 115 Leu Leu 100 Thr Gin Lys Asp Leu <210> 51 <211> 117 <212> PRT <213> Biomia tropicalis <400> 51 Gin Giu His Giu Pro Lys 1 5 Leu Leu Ile Glu Gin Ala Lys Asp Asp Phe Arg Asn Giu 10 Asn His Ile Giu Lys Gly Phe Asp His Glu His Gin Asn Lys Ser Val Cys Ala Ile Leu Tyr Lys Giu Leu Met Ile Glu Gin His Gin Glu Leu Asfl Gin Glu Lys Ile Arg Glu Leu Gly Ala Gly Ala Leu Glu Arg Leu Lys Arg WO 01/30817 PCT/AU00/01227 -71 Asp Leu Asn Ile Leu Glu Arq Phe Asn Ser Lys Ile Leu Leu Lys Asp Leu Lys 100 105 Tyr Glu Glu Ala Gin Thr Leu 90 Glu Thr Glu Gin Lys Val Lys 110 Asp Ile Gin Thr Gin 115 <210> 52 <211> 117 <212> PRT <213> Blomia tropicalis <400> 52 Gin Glu His Lys Pro Lys Lys Asp Asp 1 5 Arg Asp Glu Phe Asp His Leu Leu Ile Gin Ala Asn His Ile Glu Lys Gly Glu His Gin Leu His Tyr Leu Gin His Gin Asp Glu Leu Asn Glu Asn Lys Ser Lys Glu Leu Gin Glu Lys Ile Arg Glu Leu Val Val Cys Ala Ile Glu Gly Ala Gin Gly Ala Leu Glu Glu Leu Lys Arg Asp Leu Asn lle Glu Arg Phe Asn Glu Glu Ala Gin Thr Leu Ser Lys Ile Leu Lys Asp Leu Glu Thr Glu Gin Lys Val Lye 110 WO 01/30817 PCT/AU00/01227 -72- Asp Ile Gin Thr Lys 115 <210> 53 <211> 117 <212> PRT <213> Blomia tropicalis <400> 53 Gln Gin His Lys Pro Lys Lys Asp Asp Arg Asn Glu Phe Asp His Leu Leu Ile Glu Gin Ala Asn His Ile Glu Lys Gly Glu His Gln Asn Lys Ser Leu Leu Tyr Leu Gin His Gin Asp Glu Leu Asn Lys Glu Leu Gin Glu Lys Ile Arg Glu Leu Val Val Cys Ala Ile Glu Gly Ala Gly Ala Leu Glu Glu Leu Lys Arg Asp Leu Asn Ile Leu Glu Arg Phe Asn Glu Glu Ala Gin Thr Leu Ser Lys Ile Leu Lys Asp Leu Glu Thr Glu Gin Lys Val Lys 110 Asp Ile Gin Thr Gin 115 <210> <211> <212> WO 01/30817 WO 01/08 17PCT/AUOO/01227 73 <213> Blomia tropicalis <400> 54 Met Lys Phe Ala Ile Val Leu Ile Ala Cys Phe Ala Ala Ser Val Leu 1. 5 10 Ala <210> <211> <212> <213> 930

DNA

Blomia tropicalis <400> tttttttttt aaaaaaaaga ttttaagtta catataaaca aaaagacaaa gggaaccctt aagtaacaca tatcaggtaa cacaagccaa aaccatgcta ttgaatgaaa.

tgcgccatga aacattttgg gatttgaagg gtacaatttt tttttaacta tttttttttt aagggataaa aataaaaaag cagtaaaccc aaagacaaac aaatttaaag ccctttaaaa aatatcaaag agaaggatga tcgaaaaggg acaagagcaa tcgaaggagc aacgattcaa aaaccgaaca acatttttga taaaaaaaaa tttttttttt gaanaacaaa.

aaatagccag ctgtaaaaaa caccgacaat aactactaag ccagcatact gtgagataaa tttccgaaac agaacatcaa ggaattgcaa c caaggagc t ctacgaagag aaaagtgaag tatgattaaa aaaaaaaaaa tttttttttt aaacaaatga aactgaccta acatataaaa actaacctca aaaccaaaac acgacaatga accgtgggca gaattcgatc ttgctttact gagaaaatca ttggaacgtg gctcaaactc gatattcaaa tttttttatt caagtcctaa ccgnggccta aacatcataa gagtaaaaag aaacaaaaaa aaaatatcat actcggtttt acttgttgat tgcaacacca ttcgagaact aattgaagcg tcagcaagat cccaataaaa aaaagtaaca gtaaacaact a cc tag tat a ctcagtcaaa acataaaagg caggactaga.

a acaagatgt ggctcaagag cgaacaggca.

actcgacgaa tgatgttgtt aactgatctt cttgcttaag atttagaatt tgtcaataaa tgttcaataa ataaattcaa 900 930 <210> <211> <212> <213> 56 899

DNA

Blomia tropicalis WO 01/30817 WO 0130817PCT/AUOO/01227 74 <400> 56 ~tttttttttt tttagataat caaagataat cactttgatt ttggatttgg cattcgttct agcggtagcc cgccatcgtt gaaggatgat cgaaaaggga caagagcaag cgaaggagcc acgattcaac aaccgaacaa tttttttttt ttaatttata tagactcgtt tca tc tccag gtataggtgt tcatcaaatt ttggtttagg cttattgcct ttccgaaacg gaacatcaat gaattgcaag caaggagctt tacgaagagg aaagtgaagg gttttacaaa attaattata tgtaaacgcg taaactctcg tgccctcagc cttcaccaag aaaacactca gctttgccgc aattcgatca tgctttactt agaaaatcat tggaacgtga ctcaaactct atattcaaac ttttttgtat gttgattgat caggcaactg aatgtatttg agtaacaata cttgaatttg caatccacaa ttcggtt ttg cttgttgatc gcaacaccaa tctagaactt attgaagcga cagcaagatc ccaataaaaa aaatttaata tgatatttat aca ttg ttgc atctccaaat gacttgacct gttgtttgtg actcaaacaa gctcaagagc gaacaggcaa ctcgacgaat gatgttgttt actgatctta ttgcttaagg tttagaattg ataaattaga catacataat agattgaggt ctttgtcttt tttttccatc tt ttgaaggg caatgaagt t acaagccaaa accatgctat tgaatgaaaa gcgccatgat ac at tttgga at ttgaagga tacaatttta catttttgat atgattaaat gtcaataaat gttcaataaa taaaaaaaaa aaaaaaaaa 899 <210> 57 <211> 923 <212> DNA <213> Blomia tropicalis <400> 57 gtcgtgcgct agcaaacatg tcggctgctg tatatatata tgatgataat aatgtzgtcat gtttcgatgt ttcttattgc atttccgaaa gagaacatca aggaattgca cccaaggagc actacgaaga gcacactaaa gatggatcgt gttgcagtat tatatttaaa atatgtattli ccggtcaatg ctaaaacact ctgctttgcc cgaattcgat attgctttac agagaaaatc t ttggaacgt ggctcaaact tgtttggcaa tatactacga gatctatacc gtaaaataat tacgtgtgta tgtatgtttt cacaatccac gcttcggttt cacttgttga ttgcaacacc attcgagaac gaattgaagc ct cagc aaga ctcaaaacta actcatctga gaacctagca tatatatatg tacacttgtg tggaaagata aaactcaaac tggctcaaga tcgaacaggc aactcgacga ttgatgttgt gaactgatct tcttgcttaa tacacaattt ctaggctaat caatggccgc taaaacaaat tacggataaa tctgtttttt aacaatgaag gcacaagcca aaaccatgct attgaatgaa ttgcgccatg taacattttg ggatttgaag ggcaactagt aactgcactg cggttcagca taaaacccta tggtgaaaac tgatgcgctt ttcgccatcg aagaaggatg atcgaaaagg aacaagagca atcgaaggag gaacgat Lca gaaaccgaac WO 01/30817 WO 0130817PCT/AUOO/O1 227 75 aaaaagtgaa ggatattcaa acccaataaa aatttagaat tgtacaattt tacatttttg 840 atatgattaa atgtcaataa atgttcaata aataaattca atttttaact ataaaaaaaa 900 aaaaaaaaaa aaaaaaaaaa aaa 923 <210> 58 <211> 892 <212> DNA <213> Blomia tropicalis <400> 58 taaacgaaaa gtcaaattta tcaatttgtg actttgtcca ttaagattag gtcacttgaa tgtccacaca cttcggtttt acttgttgat tgcaacacca ttcgagaact aattgaagcg tcagcaagat cccaataaaa tgttcaataa tatggtcatg ttatatgtta gcaatggtgc aatcggcatc aatcgttatg tatcttccgg aactcaaaca ggctcaagag cgaacaggca actcgacgaa tgatgttgtt aactgatctt cttgcttaag atttagaatt ataaattcaa tttattcatt ttttttcaaa ttcaatcacc ctcaggcaac ttcactcttg cttaaagtct acaatgaagt cacaagccaa aaccatgcta ttgaatgaaa tgcgccatga aacattttgg gatttgaagg gtacaatttt tttttaaaaa atcaactcaa gtgatcggaa aatctaccat gagtaacgat gcattgatga ggcaaatcga tcgccatcgt agaaggatga tcgaaaaggg acaagagcaa tcgaaggagc aacgattcaa aaaccgaaca acatttttga aaaaaaaaaa aagtttctat tctccttgtc cggttt tcaa agttaaagac ttatttcacc tttcaatctt tcttattgcc tttccgaaac agaacatcabi ggaattgcaa c caaggagc t ctacgaagag aaaagtgaag tatgattaaa aaaaaaaaaa tatctacaaa ggcctttgcc cgatgaacgg ccggttttgt tt tcgtaacg aaagattcgt tgctttgccg gaattcgatc ttgctttact gagaaaatca ttggaacgtg gctcaaactc gatattcaaa tgtcaataaa aa 210> 59 <211> 738 <212> DNA <213> Blomia tropicalis <400> 59 tttttttttt cncattttat ggcatacaaa atacggttgg tttttttttt tttttttttt tttttttttt ttttttttna caaaaagggg ttcgattgat tgggcaaata gagtcattta catcgatttg ggtttggggg 120 ctgaacgatg acgagcccga ccaggacgac gacgacgttt atcgttgtcc 180 tttgtttagg aaaacactca caatccacaa actcaaacaa caatgaagtt 240 WO 01/30817 WO 0130817PCT/AUOO/01227 76 cgccatcgtt ga aggatga t cgaaaaggga caagagcaag cgaaggagcc acgattcaac aaccgaacaa catttttgat aaaaaaaaaa cttattgcct ttccgaaacq gaacatcaat gaattgcaag caaggagctt tacgaagagg aaagtgaagg atgattaaat aaaaaaaa gctttgccgc aattcgatca tgctttactt agaaaatcat tggaacgtga ctcaaactct atattcaaac gtcaataaat ttcggttttg cttqttgatc gcaacaccaa tcgagaactt attgaagcga cagcaagatc ccaataaaaa gttcaataaa gctcaagagc qaacaggcaa ctcgacgaat gatgttgttt acigatctta ttgcttaagg tttagaattg taaattcaat acaagccaaa accatgctat tgaatgaaaa gcgccatgat acattttgga atttgaagga tacaatttta ttttaactat <210> <211> 557 <212> DNA <213> Blomia tropicalis <400> tccttgttta ttcttattgc atttccgaaa gagaacatca aggaattgca cccaaggagc actacgaaga aaaaagtgaa atatgattaa aaaaaaaaaa ggaaaacact ctgctttgcc cgaattcgat attgctttac agagaaaatc tttggaacgt ggctcaaact ggatattcaa atgtcaataa aaaaaaa ctctatccac gcttcggttt cacttgttga ttgcaacacc attcgagaac gaattgaagc ct cagcaaga acccaataaa atgttcaata aaactcaaac tggctcaaga t cgaacaggc aactcgacga ttgatgttgt gaactgatct tcttgcttaa aatttagaat aataaattca aacaatgaag gcacaagcca aaaccatgct attgaatgaa ttgcgccatg taacattttg ggatttgaag tgtacaattt atttttaaaa ttcgccatcg aagaaggatg at cgaaaagg aacaagagca atcgaaggag gaacgattca gaaaccgaac tacatttttg aaaaaaaaaa <210> 61 <211> 520 <212> DNA <213> Blomia tropicalis <400> 61 aaaacactca caatccacaa actcaaacaa caatgaagtt cgccatcgtt cttattgcct gctttgccgc ttcggttttg gctcaagagc acaagccaaa gaaggatgat ttccgaaacg 120 aattcgatca cttgttgatc gaacaggcaa accatgctat cgaaaaggga gaacatcaat 180 WO 01/30817 WO 0130817PCT/AUOO/01227 77 tgctttactt ~aaaatcat tggaacgtga ctcaaactct atattcaaac gtcaataaat gcaacaccaa tcagaactt attgaagcga cagcaagatc ccaataaaaa gttcaataaa ctcgacgaat QatQttqttt actgatctta ttgcttaagg tttagaattg taaaaaaaaa tgaatgaaaa gcqccatgat acattttgga atttgaagga tacaatttta aaaaaaaaaa caagagcaag gaattgcaag cgaaggagcc caaggagctt acgattcaac tacgaagagg aaccgaacaa aaagtgaagg catttttgat atgattaaat <210> 62 <211> 523 <212> DNA <213> Blomia tropicalis <400> 62 ttaggaaaac tgcctgcttt aaacgaattc tcaattgctt gcaagagaaa agctttggaa agaggctcaa gaaggatatt taaatgtcaa actcacaatc gccgcttcgg gatcacttgt tacttgcaac atcattcgag cgtgaattga actctcagca caaacccaat taaatgttca cacaaactca t tt tggctca tgatcgaaca accaactcga aacttgatgt agcgaactga agatcttgct aaaaatttag ataaaaaaaa aacaacaatg agagcacaag ggcaaaccat cgaattgaat tgtttgcgcc tcttaacatt taaggatttg aattgtacaa aaaaaaaaaa aagttcgcca c caaaga agg gctatcgaaa gaaaacaaga atgatcgaag t tggaacgat aaggaaaccg ttttacattt aaa tcgttcttat atgatttccg agggagaaca gcaaggaatt gagcccaagg tcaactacga aacaaaaagt ttgatatgat <210> 63 <211> 521 <212> DNA <213> Blomia tropicalis <400> 63 aaaacactca gctttgccgc aattcgatca tgctttactt agaaaatcat tggaacgtga ctcaaactct caatccacaa ttcggttttg cttgttgatc gcaacaccaa tcgagaactt attgaagcga cagcaagatc actcaaacaa gctcaagagc gaacaggcaa ctcgacgaat gatgttgttt actgatctta t tgcttaagg caatgaagtt acaagccaaa accatgctat tgaatgaaaa gcgccatgat acattttgga atttgaagga cgccatcgtt gaaggatgat cgaaaatgga caagagcaag cgaaggagcc acgattcaac aaccgaacaa cttattgcct ttccgaaacg gaacatcaat gaattgcaag caaggagctt tacgaagagg aaagtgaagg WO 01/30817 WOOI/0817PCT/AUOO/01227 78 atattcaaac ccaataaaaa tttagaattg tacaatttta catttttgat atgattaaat 480 gtcaataaat Qttcaataaa taaattcaat ttttaactat a 521 <210> 64 <211> 509 <212> DNA <213> Blomia tropicalis <400> 64 tgcctgcttt aaacgaattc tcaattgctt gcaagagaaa agctttggaa agaggctcaa gaaggatatt taaatgtcaa aaaaaaaaaa gccgcttcgg gatcacttgt tacttgcaac atcattcgag cgtgaattg& actctcagca caaacccaat taaatgttca aaaaaaaaaa ttttggctca tgatcgaaca accaactcga aacttgatgt agcgaactga agatcttgct aaaaatttag ataaataaat aaaaaaaaa agagcacaag ggcaaaccat cgaat tgaat tgtttgcgcc tcttaacatt taaggat ttg aattgtacaa tcaattttta ccaaagaagg gctatcgaaa gaaaacaaga atgatcgaag ttggaacgat aaggaaaccg ttttacattt actataaaaa atgatttccg agggagaaca gcaaggaat t gagcccaagg tcaactacga aacaaaaagt ttgatatgat aaaaaaaaaa <210> <211> 461 <212> DNA <213> Biomia tropicalis <400> ctgctttgcc cgaattcgat attgctttac agagaaaatc tttggaacgt ggctcaaact ggatattcaa atgtcaataa gcttcggttt cacttgttga ttgcaacacc attcgagaac gaattgaagc ctcagcaaga acccaataaa atgttcaata tggctcaaga tcgaacaggc aactcgacga ttgatgttgt gaactgatct tcttgcttaa aatttagaat aataaaaaaa gcacaagcca aaaccatgct attgaatgaa ttgcgccatg taacattttg ggatttgaag tgtacaattt aaaaaaaaaa aagaaggatg atcgaaaagg aacaagagca atcgaaggag gaacgattca gaaaccgaac tacatttttg a atttccgaaa gagaacatca aggaattgca cccaaggagc actacgaaga aaaaagtgaa atatgattaa <210> 66 WO 01/30817 WO 0130817PCT/AUOO/01227 79 <211> 434 <212> DNA <213> Blomia tropicaliB <400> 66 gatt tccgaa ggagaacatc aaggaattgc gcccaaggag aactacgaag caaaaagtga gatatgatta aaaaaaaaaa acgaattcga aattgcttta aagagaaaat ctttggaacg aggctcaaac aggatattca aatgtcaata aaaa tcacttgttg cttgcaacac cattcgagaa tgaattgaag tctcagcaag aacccaataa aatgttcaat atcgaacagg caactcgacg cttgatgttg cgaactgatc atcttgctta aaatttagaa aaataaattc caaaccatgc aattgaatga tttgcgccat ttaacatttt aggatttgaa ttgtacaatt aatttttaac tatcgaaaag aaacaagagc gatcgaagga ggaacgatt c ggaaaccgaa ttacattttt tataaaaaaa <210> 67 <211> 340 <212> DNA <213> Blomia tropicalis <400> 67 actagacttg gaaaatcatt ggaacgtgaa tcaaactctc tattcaaacc tcaataaatg caacaccaac cgagaac ttg ttgaagcgaa agcaagatct caataaaaat ttcaataaat tcgacgaatt atgttgtttg ctgatcttaa tgcttaagga ttagaattgt aaaaaaaaaa gaatgaaaac cgccatgatc cattttggaa tttgaaggaa acaattttac aaaaaaaaaa aagagcaagg aattgcaaga gaaggagccc aaggagcttt cgattca&ct acgaagaggc accgaacaaa aagtgaagga atttttgata tgattaaatg <210> 68 <211> 134 <212> PRT <213> Blomia tropicalis <400> 68 Met Lys Phe Ala Ile Val Leu Ile Ala Cys Phe Ala Ala Ser Val Leu 1 5 10 1s WO 01/30817 WO 0130817PCT/AUOO/01227 Ala Gin Giu His Leu Leu Lys Pro Lys Lys Asp Phe Arg Asn Glu Phe Asp Gly Giu His Ile Giu Gin Ala His Ala Ile Giu Gin Leu so Leu*Tyr Leu Gin His Gin Leu Asp Glu Asn Glu Asn Lys Ser Lys Giu Leu Gin Lys Ile Ile Leu Leu Asp Val Val Ala Met Ile Giu Gly Ala Gin Gly Ala Glu Arg Giu Leu Lys Arg Thr Asp Leu Leu Ser Lys 115 Ile Leu Giu Arg Asn Tyr Giu Giu Ala Gin Thr 110 Gin Lys Val Ile Leu Leu LYS Leu Lys Giu Thr Lys Asp 130 Ile Gin Thr Gin <210> 69 <211> 134 <212> PRT <213> Biomia tropicalia <400> 69 met Lys Phe Ala Ilie Val 1 5 Leu Ile Ala Phe Ala Ala Ser Val Leu Ala Gin GiU His Lys Pro Lys Lys Asp 25 Asp Phe Arg Asn Glu Phe Asp His Leu Leu Ilie Glu Gin Ala Asn His Ala le Giu Asn Gly Giu His WO 01/30817 PCT/AU00/01227 -81 Gln Leu Ser Lys Tyr Leu Gin His Leu Asp Glu Glu Asn Lye Glu Leu Gin Lys Ile Ile Arg Asp Val Val Met Ile Glu Ala Gin Gly Ala Glu Arg Glu Leu Lys Arg Thr Asp Leu Leu Ser Lys 115 Lys Asp Ile 130 Leu Glu Arg Tyr Glu Glu Ala Gin Thr 110 Gin Lys Val Leu Leu Lys Lys Glu Thr Gln Thr Gln <210> <211> 127 <212> PRT <213> Blomia tropicalis <400> Asp Lys Thr Val Gly Asn 1 5 Lys Asp Asp Phe Arg Asn Ser Val Leu Glu Phe Asp Gly Glu His Gin Glu His Lys Pro Lys Leu Leu Ile Asn His Ala Gin Leu Asp Glu Lys 4 Asn Lys Gin Leu Leu Ser Lys Glu Glu Gin Ala Leu Gin His Gin Glu Lys Glu Leu Asn WO 01/30817 WO 0130817PCT/AUOO/01227 82 Ile Arg Giu Leu Val Val Cys Ala Ile Glu Gly Ala Gly Ala Leu Giu Giu Leu Lys Arg Asp Leu Asn Ile Leu Giu Arg Phe Asn Asp Leu Lys 115 Glu Giu Ala Gin Leu Ser Lys Ile Leu Leu Lys 110 Giu Thr Giu Gin Val Lys Asp Ile Gin Thr Gin 125 <210> 71 <211> 84 <212> PRT <213> Biomia tropicalis <400> 71 Leu Asp Leu Gin His Gin Leu Asp Giu Asn Glu Asn Lys Ser Lys Giu Leu Gin Lys Ile Ile Arg Giu Leu Asp Val Val Cys Ala Met Arg Thr Asp Ile Giu Gly Ala Gin Gly Ala Leu Giu Arg Giu Leu Leu Ann Ile so Leu Giu Arg Asri Tyr Giu Giu Gin Thr Leu Ser Ilie Leu Leu Lys Leu Lys Giu Thr Gin Lys Val Lys Ile Gin Thr Gin WO 01/30817 WO 0130817PCT/AUOO/01227 -83- <210> 72 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer 4400> 72 cccggatcca caatgaagtt cgccatcgtt ctt 33 <210> 73 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:primer <400> 73 gctctagatt attggtttga atatc <210> 74 <211> 507 <212> DNA <213> Blomia tropicalis <220> <221> CDS <222> <400> 74 ggccagag cac ccc gct caa gct ctc gac ttt acc agc tgt gcc cgg atg His Pro Ala Gin Ala Leu Asp Phe Thr Ser Cys Ala Arg Met 1 S WO 01/30817 WO 0130817PCT/AUOO/01227 84gat gga gct ctg gga Asp Gly Ala Leu Gly gcc aag gta gct Ala Lys Val Ala gct gcc tgc atc Ala Ala Cys Ile tcg 98 Ser ggt 146 Gly agt tgc aag ttt Ser Cys Lys Phe aac tgt ggc Asn Cys, Gly gtc tgt tct Val Cys Ser acg Thr tgt gag agg Cys Glu Arg gga cgt ccr.

Gly Arg Prc tgg ccc aat Trp Pro Asr gtgacacact ccatcaattt ttgtggcttt aataattttt aaaaaaaaaa cgc tgt ggc aac ggt ggc ggt gaa 194 Arg Cys Gly Asn Gly Gly Gly Glu ctg ccc tcc agg ggt taattatttt Leu Pro Ser Arg Gly atttatatat gctttcaata ggaataacct ttgacaattt caaaattaat taaggtttta atttttttaa aattatccga gatgtttgac taaatttaaa aattgtttta taaataaaaa aaaattcgt gtcatttggt. ctgtgaaagt 248 tcaattttgg tcacactttc 308 ttttaaagtt catcaatttt 368 agagacaatt gattaattaa 428 ttttttattt ttggaaaaaa 488 507 <210> <211> <212> PRT <213> Blomia tropicalis <400> His Pro Ala Gin Ala Leu Asp Phe Thr Ser Cys Ala Arg Met Asn Asp 1 5 10 Gly Ala Leu Gly Ala Lys Val Ala Gln Ala Ala Cys Ile Ser Ser Cys WO 01/30817 WO 0130817PCT/AUOO/01227 Lys Phe Gin Asn Cys Gly Thr Gly H-is Cys Glu Arg Arg 40 Gly Gly Arg Glu Trp Pro Pro Thr Cys Val Cys so Ser Arg Cys Gly Asn Gly Gly Gly 55 Leu Pro Ser Arg <210> 76 <211> 26 <212> DNA <213> Blomia tropicalis <400> 76 cgggatccgc tctcgacttt accagc <210> 77 <211> 26 <212> DNA <213> Blomia tropicalis <400> 77 cggaattctt aacccctgga gggcag <210> 78 <211> <212> DNA <213> Blomia tropicalis <400> 78 gaaaagagct ctcgacttta ccagc WOO01/30817 PCT/AUOO/01227 86 <210> 79 <211> 26 <212> DNA <213> Blomia tropicalis <400> 79 cggaattctt aacccctgga gggcag 26

Claims (28)

1. An isolated nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding a protein allergen from Blomia tropicalis wherein said protein allergen is selected from Btl1, BtlO and BtA2, each as herein defined, and polymorphic variants thereof and a polymorphic variant of Bt5 as herein defined.

2. The nucleic acid molecule of Claim 1 comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a nucleotide sequence encoding Btll having an amino acid sequence as set forth in any one of SEQ ID NO: 2 to SEQ ID NO:14.

3. The nucleic acid molecule of Claim 2 comprising a nucleotide sequence encoding Btl wherein said nucleotide sequence is as substantially set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13, or is capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13 or a complementary form thereof under medium stringency conditions. i4. The nucleic acid molecule of Claim 2 comprising a nucleotide sequence encoding a polymorphic variant of Btl 1 comprising a nucleotide sequence substantially as set forth in SEQ ID NO:27.

5. The nucleic acid molecule according to Claim 1 comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a nucleotide sequence encoding Btl0 having an amino acid sequence as set forth in SEQ ID NO:29 or SEQ ID NO:31

6. The nucleic acid molecule of Claim 5 comprising a nucleotide sequence encoding BtlO wherein said nucleotide sequence is as substantially as set forth in SEQ ID NO:28 or SEQ ID NO:30 or a nucleotide sequence capable of hybridizing to SEQ ID NO:28 or SEQ ID NO:30 or a complementary form thereof under medium stringency conditions. P.QPEREjE#jmdcd claims\2512433 ecrcod dainsdoc. I SdO -86-

7. The nucleic acid molecule of Claim 6 comprising a nucleotide sequence encoding a polymorphic variant of Bti0.

8. The nucleic acid molecule of Claim 1 comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a nucleotide sequence encoding a polymorphic variant of Bt5 having an amino acid sequence as set forth in SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71.

9. The nucleic acid molecule of Claim 8 comprising a nucleotide sequence encoding a polymorphic variant of Bt5 wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:38 to SEQ ID NO:40 or SEQ ID NO:55 to SEQ ID NO:67 or a nucleotide sequence capable of hybridizing to SEQ ID NO:38 to SEQ ID NO:40 or SEQ ID NO:55 to SEQ ID NO:67 or a complementary form thereof under medium stringency conditions. The nucleic acid molecule of Claim 1 comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a nucleotide sequence encoding BtA2 having i: i an amino acid sequence as set forth in SEQ ID

11. The nucleic acid molecule of Claim 10 comprising a nucleotide sequence encoding BtA2 wherein said nucleotide sequence is substantially as set forth in SEQ ID NO:74 or a nucleotide sequence capable of hybridizing to SEQ ID NO:74 under medium stringency condition.

12. The nucleic acid molecule of Claim 10 or 11 comprising a nucleotide sequence o encoding a polymorphic variant of BtA2.

13. An isolated cDNA molecule comprising a sequence of nucleotides as set forth in SEQ ID NO:1.

14. An isolated cDNA molecule comprising a sequence of nucleotides as set forth in SEQ ID NO:2. P %DPEREjh\Ejh\&ncwdd dwMA2512433 accd damdecQIOM -87- 1 C A X ,lc, T A c on\ nn r r o~c)frh; I 11 I~I tCU IIIV~U~A.I%. %AJVII jJL.IIII a ~Jc u~iI%. VIi IIvvru l ,rr rrrl l SEQ ID NO:13.

16. An isolated cDNA molecule comprising SEQ ID NO:27.

17. An isolated cDNA molecule comprising SEQ ID NO:28.

18. An isolated cDNA molecule comprising SEQ ID a sequence of nucleotides as set forth in a sequence of nucleotides as set forth in a sequence of nucleotides as set forth in S*

19. An isolated cDNA molecule comprising a sequence of nucleotides as set forth in SEQ ID

20. An isolated cDNA molecule comprising a sequence of nucleotides as set forth in SEQ ID NO:56.

21. An isolated cDNA molecule comprising a sequence of nucleotides as set forth in SEQ ID NO:57.

22. An isolated cDNA molecule comprising a sequence of nucleotides as set forth in SEQ ID NO:58.

23. An isolated cDNA molecule comprising a sequence of nucleotides as set forth in SEQ ID NO:59.

24. An isolated cDNA molecule comprising a sequence of nucleotides as set forth in SEQ ID P:\OPER\Ejh'Eh\acndodlclimSI2433 woted claims docc. I AW8A -88- An isolated SEQ ID NO:61.

26. An isolated SEQ ID NO:62.

27. An isolated SEQ ID NO:63.

28. An isolated SEQ ID NO:64.

29. An isolated SEQ ID An isolated SEQ ID NO:66.

31. An isolated SEQ ID NO:67.

32. An isolated SEQ ID NO:74. cDNA molecule comprising a sequence of nucleotides as set forth in cDNA molecule comprising a sequence of nucleotides as set forth in cDNA molecule comprising a sequence of nucleotides as set forth in cDNA molecule comprising a sequence of nucleotides as set forth in cDNA molecule comprising a sequence of nucleotides as set forth in cDNA molecule comprising a sequence of nucleotides as set forth in cDNA molecule comprising a sequence of nucleotides as set forth in cDNA molecule comprising a sequence of nucleotides as set forth in 9* 9* 9 *r 9* 9 9 9. 9. 9 9

99.9 .9 9 99*** 33. An isolated protein allergen from Blomia tropicalis wherein said protein allergen is selected from Btl 1, BtlO and BtA2, each as herein defined, polymorphic variants thereof and a polymorphic variant of Bt5 as herein defined. 34. The isolated protein of Claim 33 comprising an amino acid sequence defining Btl 1, said amino acid sequence substantially as set forth in SEQ ID NO:3 to SEQ ID NO:12 or SEQ ID NO:14. P \OPER\Ejh\Ejh~amcndd claimN 3 cffcucd caims doc-10 S -89- The isulated protein of Claim 34 comprising an amino acid sequence defining Btl 1, said amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO: 13 or a nucleotide sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:13 or a complementary form thereof under medium stringency conditions. 36. The isolated protein of Claim 33 comprising an amino acid sequence defining BtlO, said amino acid sequence substantially as set forth in SEQ ID NO:28 or SEQ ID NO:31. 37. The isolated protein of Claim 36 comprising an amino acid sequence defining BtlO, said amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:28 or SEQ ID NO:30 or a nucleotide sequence capable of hybridizing to SEQ ID NO:28 or SEQ ID NO:30 or a complementary form thereof under medium stringency conditions. 38. The isolated protein of Claim 33 comprising an amino acid sequence defining a polymorphic variant of Bt5, said amino acid sequence substantially as set forth in SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71 or an antigenic derivative thereof or having at least about 45% similarity to an amino acid sequence set forth in SEQ ID NO:49 to SEQ ID NO:53 or SEQ ID NO:68 to SEQ ID NO:71. 39. The isolated protein of Claim 38 comprising an amino acid sequence defining a polymorphic variant of Bt5, said amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:38 to SEQ ID NO:40 or SEQ ID NO:55 to SEQ ID NO:67 or a nucleotide sequence capable of hybridizing to SEQ ID NO:38 to SEQ ID NO:40 or SEQ ID NO:55 to SEQ ID NO:67 or a complementary form thereof under medium stringency conditions. The isolated protein of Claim 33 comprising an amino acid sequence defining BtA2, said amino acid sequence substantially as set forth in SEQ ID P.ZOPeR\Ejh\Ejhva m clainsN2 12433 ow ed claims 41. The isolated protein of Claim 40 comprising an amino acid sequence defined BtA2, said amino acid scqucnce encoded by the nucleotide sequence set forth in SEQ ID NO:74 or a nucleotide sequence capable of hybridizing to SEQ ID NO:74 or a complementary form thereof under medium stringency conditions. 42. An isolated protein comprising the amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:3 to SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14. 43. An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:29 or SEQ ID NO:31. 44. An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:49. An isolated protein comprising the amino acid sequence set forth in SEQ ID 46. An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:52. 47. An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:53. 48. An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:54. a oooo a a a a 6 a a a* a a a a a o o* a 49. An isolated protein comprising the amino acid sequence set forth in SEQ ID An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:56. P.%OPEREjjtacd& cid climNS12433 corcd dsinmsd-IOM NO:57 52. NO:58 53. NO:59 54. NO:61 56. NO:62 57. NO:63 58. NO:64 59. NO:66 -91 An isolated protein comprising the aimino acid sequence sfrth in SE..Q D An isolated protein comprising the amino acid sequence set forth in SEQ ID An isolated protein comprising the amino acid sequence set forth in SEQ ID An isolated protein comprising the amino acid sequence set forth in SEQ ID An isolated protein comprising the amino acid sequence set forth in SEQ ID An isolated protein comprising the amino acid sequence set forth in SEQ ID An isolated protein comprising the amino acid sequence set forth in SEQ ID An isolated protein comprising the amino acid sequence set forth in SEQ ID An isolated protein comprising the amino acid sequence set forth in SEQ ID An isolated protein comprising the amino acid sequence set forth in SEQ ID a a a. a a a apa a a. a P:%OPeRjh\jh~mmdco clai.NsIU512433 d chumsd=40)OW5 -92- 61. An isolated protein comprising the amino acid sequence set forth in SEQ ID T r, 62. An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:68. 63. An isolated protein comprising the amino acid sequence set forth in SEQ ID NO:69. 64. An isolated protein comprising the amino acid sequence set forth in SEQ ID An isolated protein comprising the amino acid sequence set forth in SEQ ID 66. A method of preventing, reducing or otherwise ameliorating a B. tropicalis hypersensitivity condition in a subject, said method comprising administering to said subject an effective amount of an allergen from said B. tropicalis for a time and under conditions sufficient to desensitize said individual. 67. A method of modulating, in a subject, an immune response directed to B. tropicalis allergen, said method comprising administering to said subject an effective amount of said allergen for a time and under conditions sufficient to up-regulate, down-regulate or otherwise modulate said immune response. 68. A method for the prophylactic treatment of an allergic condition comprising the step of administering to an individual a nucleic acid molecule comprising a gene encoding Btl 1 whereby airway hyper-reactivity or airway inflammation is prevented. 69. The method of Claim 68 wherein the allergic condition is allergic asthma, atopic dermatitis and/or rhinitis. S S S S a. S a S S S. S S 5, PAOPEREjMELmcndolimN25i2433 cmad claims dc-IOMVS -93- The method of Claim 6 or 69 herin, the nucleic acid molecule is in the form of an eukaryotic expression vector. 71. The method of Claim 68 or 69 or 70 wherein the eukaryotic expression vector is administered in a pharmaceutical composition comprising a carrier selected from the group consisting of normal saline and a liposome. 72. The method of Claim 68 wherein the individual is a human. 73. The method of Claim 68 or 69 or 70 wherein the vector is a plasmid vector. 74. The method of Claim 68 wherein the administration is via intramuscular injection. Use of a B. tropicalis allergen in the manufacture of a medicament for modulating an immune response. 76. An agent when used for modulating an immune response, said agent comprising a B. tropicalis allergen. 77. A composition when used in modulating an immune response comprising a B. tropicalis allergen and one or more pharmaceutically acceptable carriers and/or diluents. 78. An isolated antibody to a B. tropicalis allergen. *79. A method for detecting antibody directed to a B. tropicals allergen in a biological sample from a subject, said method comprising contacting said biological sample with B. tropicalis allergen or an antigenic derivative thereof for a time and under conditions sufficient for an antibody-protein complex to form, and then detecting said complex. P %PE~~jhEjhsmcdcdclainmt%2512413 wrreed claimsdmc.I 94 A pharmaceutical composition when used for modulating an immune response directed to irupicalis., said cmoioncomprLISAIng W-1legnfrmB tsrirldis nd one or more pharmaceutically acceptable carriers and/or diluents. DATED this 10Oth day of August, 2005 CHUA, Kaw, Van CHEONG, Nge LEE, Bee, Wah by its Patent Attorneys DAVIES COLLISON CAVE