WO2003016529A1

WO2003016529A1 - Novel blomia tropicalis antigens and uses therefor

Info

Publication number: WO2003016529A1
Application number: PCT/AU2002/001125
Authority: WO
Inventors: Kaw Yan Chua; Nge Cheong; Bee Wah Lee
Original assignee: Kaw Yan Chua; Nge Cheong; Bee Wah Lee
Priority date: 2001-08-20
Filing date: 2002-08-20
Publication date: 2003-02-27
Also published as: MY140636A; AUPR713201A0

Abstract

The present invention relates generally to a novel protein molecule and to derivatives, homologs, analogs, 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 a genetic sequence encoding said protein molecule and derivatives, homologs, analogs, chemical equivalents and mimetics thereof. The present invention further provides genetic vaccines and other compositions comprising nucleic acid molecules. The protein molecule of the present invention may be regarded as a protein allergen and used alone or in combination with known allergens provides the possibility for developing more comprehensive tests for potential allergic responses. The protein allergen or its encoding sequence of the present invention are also useful, inter alia, in a range of therapeutic, prophylactic and diagnostic applications.

Description

NOVEL BLOMIA TROPICALIS ANTIGENS AND USES THEREFOR

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Bibliographic details of references provided in the subject specification are listed at the end of the specification.

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

The domestic mite, Blomia tropicalis (Bio or Bt) classified under the family

Glycyphagidae (Liebers et al, Clin. Exp. Allergy 26: 494-516, 1996), is a main component of house dust in the tropical and subtropical regions. It has been well documented that this mite is an important triggering factor for allergic asthma and rhinitis in the tropics (Mariana et al, Southeast Asian J. rop. Med. Public Health 27(2): 392-395, 1996; Puerta et al, J. Allergy Clin. Immunol. 28(5)(1): 932-937, 1996; Puerta et al, J. Allergy Clin. Immunol. 97(2): 689-691, 1996; Arlian et α/., J. Allergy Clin. Immunol. 91(5): 1042-1050, 1993; Fernandez-Caldas et al, J. Investig. Allergol. Clin. Immunol. 7(5): 402-404, 1997; Caraballa et al, Clin. Exp. Allergy 24: 1056-1060, 1993). To date, cDNA clones coding for three allergens have been characterized from this mite. These are Bio t5 (Arruda et al, Int. Arch. Allergy Immunol. 107: 456-457, 1995; Caraballo et al, J. Allergy Clin. Immunoo. 98(3): 573-579, 1996), Bio t6 (which is now classified as Bio tl3) (Caraballo et al, J. Allergy Clin. Immunol. 97(1)(3): 420, 1996; Caraballo et al, Int. Arch. Allergy Immunol 112: 341-347, 1997) and Bio tl la (which is now classified as Bio tl2) (Puerta et al, 1996, supra). The cDNA coding for Bio t5 is 522 bp in length, containing a 432 bp open reading frame. The amino acid sequence showed approximately 40% sequence homology to Der p5 (Arruda et al, 1995, supra; Caraballo et al, 1996; supra; Lin et al, J. Allergy Clin. Immnol. 94: 989-996, 1994; Tovey et al, J. Exp. Med. 170: 1457-1462, 1989). The estimated frequency of IgE reactivity of Bio t5 to mite allergic sera was about 70% (Caraballo et al, 1996, supra; Arruda et al, American Journal of Respiratory and Critical Care Medicine 155: 343-350, 1997;). The nucleotide sequence of Bio tl3 has 934 bp in length with a 390 bp reading frame coding a 130-amino acid protein of 14.8 kD in molecular weight. Bio tl3 has a cytosolic fatty acid-binding protein (FABP) signature at 5- 22 amino acid residues. It shows 41.3% identity with the Sml4-FABP of Schistosoma mansoni and 36% identity with FABPs from rat, mouse, bovine and human. The frequency of IgE binding of allergic sera to Bio tl3 was 11% and normally weak. Bio tl2 consists of 582 base pairs. The sequence has an ATG start codon at position 35 to 37 and a stop codon TAA at position 467 to 469, which indicates a reading frame coding for a 144 amino acids residue protein. The 3' 116 bp non-translated region has a poly A tail, encoding a putative signal peptide of 20 residues and a 124-residue mature protein of approximately 14.2 kD. The frequency of IgE binding of sera from patients with asthma to Bio tl2 was approximately 50% (Puerta et al, 1996, supra). This published Bio tl2 gene sequence has an ATG start codon and a TAA stop codon followed by a poly A tail. Furthermore, the amino acids at the N-terminal end have a positively charged lysine near the N terminal with the remaining amino acids being very hydrophobic, indicative of a typical leader sequence (Non Heijne, Nucleic Acids Res. 14: 4683-4690, 1986; Perlman and Halvorson, J. Mol. Biol. 167: 391-409, 1983).

Studies on the allergenicity of B. tropicalis revealed the presence multiple allergens. Crossed radioimmunoelectrophoresis (CRIE) studies identified several species-specific IgE binding-antigens in B. tropicalis (Arlian et al, 1993, supra) while immunob lotting technique was able to reveal the presence of multiple components with molecular weights ranging from 11 to 85 kilodalton (Caraballo et al, 1993, supra).

Group 1 allergens have been isolated in various mites including Dermatophagoides pteronyssinus (Der pi), Dermatophagoides farinae (Der fl) and Euroglyphus maynei (Eur ml). The nucleotide sequence in Der pi is shown to be 857 bases long and included a 69- base-long 5' proximal end sequence, a coding region for the entire mature Der pi protein of 222 amino acids with a derived molecular weight of about 25kD, an 89-base-long 3' noncoding region and a poly(A) tail of 33 residues (Chua et al, J. Exp. Med. 167: 175- 182, 1988).

A comparison of the deduced amino acid sequences reveals 78% identity among Eur ml, Der pi and Der fl. Both Eur ml and Der pi, and Eur ml and Der fl show approximately 85%) sequence identity with each other, and Der pi and Der fl approximately 82%> (Dilworth et al, Clin. Exp. Allergy 21: 25-32, 1991; Kent et al, Int. Arch Aller y Immunol. 99: 150-152, 1992).

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 allergens. 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. There is also a need to identify variants such as polymorphic variants of the allergens. The availability of a comprehensive range of allergens and their variants would greatly assist in providing more accurate diagnosis or therapy. This is particularly the case as different allergens or different polymorphic variants of the same allergen may yield different responses in different patients in clinical practice. The availability of allergens provides the clinician with the wherewithall to test for and identify a far wider range of allergic responses and thereby develop more appropriate, patient-targetted therapies.

SUMMA Y 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 NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:l), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.

A cDNA clone encoding the full length of an allergen from B. tropicalis has been isolated in accordance with the present invention. The allergen described herein is designated as " Blo tl".

In accordance with the present invention, therefore, it is proposed to exploit Bio tl in order to generate improved tools for diagnostic use and clinical immunotherapy in the treatment of dust mite allergy. Reference herein to a "dust mite" includes reference to a "storage dust mite" or a "house dust mite" or a "storage mite". Treatments for allergic responses to members of all species encompassed by the families Pyroglyphidae and Glycyphagidae may be facilitated by the use of the polymorphic variants of the present invention, by virtue of the potential for cross-reactivity between species.

The present invention provides, therefore, an isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding protein allergen from a mite and in particular B. tropicalis or a derivative, homolog or mimetic of said protein allergen.

The allergen is Bio tl. Reference to "Bio tl" includes reference to its derivatives, homologs, mimetics and polymorphic variants. The nucleic acid molecule of the present invention preferably comprises a nucleotide sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:2 or SEQ ID NO: 4 or SEQ LD NO:5 or SEQ ID NO:6 or a derivative, homolog or mimetic thereof or having at least about 55% or greater similarity to at least 10 contiguous amino acids in SEQ ID NO:3 or one of SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6. See Table 1 for a summary of the sequence identifiers.

The nucleic acid molecule even more preferably comprises a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:2, or a derivative, homolog or mimetic thereof or capable of hybridizing to SEQ ID NO:l or SEQ ID NO:2 or a complementary form thereof under low stringency conditions.

The present invention contemplates, therefore, a nucleic acid molecule or derivative thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:2 or a derivative, homolog or mimetic thereof or capable of hybridizing to SEQ ID NO:l or SEQ ID NO:2 or a complementary form thereof 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:4 or SEQ ID NO:5 or SEQ ID NO:6 or a sequence having at least about 55% similarity to at least 10 contiguous amino acids in one of SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6.

A preferred aspect of the present invention contemplates a nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:l or SEQ ID NO:2.

Still another aspect of the present invention contemplates a genomic nucleic acid molecule or derivative thereof capable of hybridizing to SEQ ID NO:l or SEQ ID NO:2, or a derivative or homolog or mimetic thereof or a or a complementary form thereof under low stringency conditions of 42°C. Still yet another further aspect of the present invention provides a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:l or SEQ ID NO:2 or a derivative thereof including a nucleotide sequence having similarity to SEQ ID NO:l or SEQ ID NO:2.

Another aspect of the present invention provides a protein having an amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:4 or one of SEQ ID NO:5 or SEQ ID NO:6 or is a derivative, homolog, analog, chemical equivalent or mimetic thereof having at least about 55% 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:4 or one of SEQ ID NO:5 or SEQ TD NO:6 or a derivative or homolog or mimetic thereof.

Another aspect of the present invention is directed to an isolated protein selected from the list consisting of:-

(i) protein allergen homolog of Bio tl from a mite or a derivative, homolog, analog, chemical equivalent or mimetic thereof;

(ii) protein allergen Bio tl from B. tropicalis or a derivative, homolog, analog, chemical equivalent or mimetic thereof;

(iii) a protein having an amino acid sequence substantially as set forth in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or a derivative, homolog or mimetic thereof or a sequence having at least about 55% similarity to at least 10 contiguous amino acids in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or

SEQ ID NO:6 or a derivative, homolog, analog, chemical equivalent or mimetic of said protein;

(iv) a protein encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:2 or a derivative or homolog thereof or a sequence encoding an amino acid sequence having at least about 55% similarity to at least 10 contiguous amino acids in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or a derivative, homolog, analog, chemical equivalent of said protein;

(v) a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:l or SEQ ID NO:2 or a derivative or homolog thereof or a complementary form thereof under low stringency conditions and which encodes an amino acid sequence substantially as set further in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or a derivative or homolog or mimetic thereof or an amino acid sequence having at least about

55%) similarity to at least 10 contiguous amino acids in SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6;

(vi) a protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or (v) in a homodimeric form; and

(vii) a protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or (v) in a heterodimeric form.

Yet another aspect of the present invention provides 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 Bio tl or a derivative, homolog, analog, mimetic or chemical equivalent thereof for a time and under conditions sufficient to desensitize said individual.

Still yet another aspect of the present invention relates to a method of modulating, in a subject, an immune response directed to B. tropicalis allergen Bio tl, said method comprising administering to said subject an effective amount of said allergen or a derivative, homolog, analog, chemical equivalent or mimetic thereof for a time and under conditions sufficient to up regulate, down regulate or otherwise modulate said immune response. The present invention further extends to the use of B. tropicalis allergen Bio tl 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 B. tropicalis allergen Bio tl.

Another aspect of the present invention provides a composition for use in modulating an immune response comprising B. tropicalis allergen Bio tl and one or more pharmaceutically acceptable carriers and/or diluents.

Still another aspect of the present invention is directed to antibodies to B. tropicalis allergen Bio tl and its derivatives, homologs, analogs, mimetics and chemical equivalents thereof.

A further aspect of the present invention contemplates a method for detecting antibody directed to all or part of B. tropicalis allergen Bio tl in a biological sample from a subject, said method comprising contacting said biological sample with said B. tropicalis allergen or a derivative, homolog, analog, chemical equivalent or mimetic thereof for a time and under conditions sufficient for an antibody-protein complex to form, and then detecting said complex.

Yet another aspect of the present invention is directed to a pharmaceutical composition useful for modulating an immune response directed to B. tropicalis, said composition comprising Bio tl from B. tropicalis or derivative, homolog, analog, chemical equivalent or mimetic thereof and one or more pharmaceutically acceptable carriers and/or diluents.

Single and three letter abbreviations used throughout the specification are defined in Table

2. TABLE1

Summary of Sequence Identifiers

TABLE 2

Single and three letter amino acid abbreviations

BRIEF DESCRIPTION OF THE DRAWINGS

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 Bio tl.

Figure 3 is a representation of the amino acid sequence (in one-letter code) of Bio tl allergen. Nucleotide sequence residues are numbered at the end of each row. The start codon and the stop codon are underlined.

Figure 4 is a representation of the nucleotide sequences alignment of Bio tl with other group 1 allergens from other mite species Euroglyphus maynei (Eurm 1), Dermatophagoides farinae (Der fl) and Dermatophagoides pteronyssinus (Der pl).

Figure 5 is a representation of the amino acid sequences alignment of Bio tl with other group 1 allergens from other mite species Euroglyphus maynei (Eurm 1), Dermatophagoides farinae (Der fl) and Dermatophagoides pteronyssinus (Der pl). Bio tl differs from other group 1 allergens in the regions A and B as indicated in the figure.

Figure 6 is a photographic representation showing SDS-PAGE recombinant fusion protein expression in E. coli and the expression in the yeast GS115.

Figure 7 is a diagrammatic representation showing the schedule of immunization.

Figure 8 is a graphical representation showing total Ig titers of five mice immunized with Bio tl assayed on ELISA plates coated with recombinant Bio tl expressed in E. coli.

Figure 9 is a graphical representation showing total Ig titers of five mice immunized with Bio tl assayed on ELISA plates coated with recombinant Bio tl expressed in Pichia pastoris. Figure 10 is a graphical representation showing total Ig titers of six Bio tl hybridoma clones.

Figure 11 is a photographic representation showing Western blot analysis using Bio tl monoclonal antibody 11F1.

Figure 12is a photographic representation showing Coomassie satining of SDS-PAGE gel showing the different fractions during antibody production.

Figure 13 is a photographic representation showing silver stained gel of the recombinant and MAb-purified native Bio tl .

Figure 14 is a graphical representation showing IgE reactivity with 63 Singaporean sera with yBlo tl , GST-Bio tl and nBlo tl .

Figure 15 is a graphical representation showing IgE reactivity of 70 Malaysian sera with bBlo tl, GST-Bio tl and nBlo tl.

Figure 16 is a graphical representation showing IgE reactivity of 40 adult rhinitis sera with yBlo tl, GST-Bio tl and GST alone.

Figure 17 is a graphical representation showing IgE reactivity of 40 adult asthma sera with yBlo tl, GST-Bio tl and GST alone. Sera with high IgE titer were selected for the experiment.

Figure 18 is a graphical representation showing IgE reactivity of 40 sera from asthmatic children with yBlo tl, GST-Bio tl and GST alone. Sera with high IgE titer were selected for the experiment. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel protein from the domestic dust or storage mite or a derivative, homolog, analog, 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.

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.

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 response. In a preferred embodiment, the immune response comprises a humoral response component and, most particularly, an IgE response.

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 allergen 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 a preferred embodiment, the present invention provides a nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a nucleotide sequence encoding, an amino acid sequence substantially as set forth in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or a derivative, homolog or mimetic thereof or having at least about 55% or greater similarity to at least 10 contiguous amino acids in SEQ ID NO:3 or one of SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6. The protein is referred to herein as "Bio tl". The corresponding gene is "Bio tl ". Reference to Bio tl or Bio tl includes all mutants, derivatives, homologs, mimetics and polymorphic variants.

The term "similarity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Wliere 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 (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) 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 "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 (i.e. gaps) of about 10% 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. (Nucl. Acids Res. 25: 3389, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. "Current Protocols in Molecular Biology", John Wiley & Sons Inc., Chapter 15, 1994-1998).

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-by- nucleotide 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 (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Nai, Leu, He, 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 (i.e. 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.

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°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. 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 T_m = 69.3 + 0.41 (G+C)%> (Marmur and Doty, J. Mol. Biol. 5: 109, 1962). However, the T_m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur. J Biochem. 46: 83, 1974). 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 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°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:l, or a derivative, homolog or mimetic thereof or capable of hybridizing to SEQ ID NO:l under low stringency conditions.

Preferably, the present invention contemplates a nucleic acid molecule or derivative thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:2 or a derivative, homolog or mimetic thereof or capable of hybridizing to SEQ ID NO:l or SEQ ID NO:2 or a complementary form thereof 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:4 or SEQ ID NO:5 or SEQ ID NO:6 or a sequence having at least about 55% similarity to at least 10 contiguous amino acids in SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6.

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

Without limiting the present invention to any one theory or mode of action, the protein Bio tl 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 the protein Bio tl 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. Bio tl is defined by the amino acid sequence set forth in SEQ ID NO:5 or SEQ LO NO:6. The cDNA sequence for Bio tl is defined by the nucleotide sequence set forth in SEQ ID NO:l. The nucleic acid molecule encoding Bio tl 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:l or SEQ ID NO:2, or a derivative or homolog or mimetic thereof or a complementary form thereof, under low stringency conditions of 42°C.

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

The protein and/or gene is preferably from B. tropicalis. However, the 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.

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 Bio tl. 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 residues 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 Bio tl or Bio tl should be understood as molecules exhibiting any one or more of the functional activities of Bio tl or Bio tl 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 Bio tl include fragments having particular epitopes of parts of the entire Bio tl protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.

Analogs of Bio tl 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 analogs.

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 molecules. The derivatives of the nucleic acid molecules of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in co- suppression 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 NaBH₄; 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 NaBH₄.

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 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or 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-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, 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/AU97/00668 [International

Patent Publication No. WO 97/15663].

Crosslinkers can be used, for example, to stabilize 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH )_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.

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 Bio tl is a cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:l or SEQ ID NO:2 or a derivative thereof including a nucleotide sequence having similarity to SEQ ID NO:l or SEQ ID NO:2.

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 SEQ ID NO:l or SEQ ID NO:2 under low stringency conditions. Preferably, low stringency is at 42°C.

The nucleic acid molecule may be ligated to an expression vector capable of expression in a prokaryotic cell (e.g. E.coli) or a eukaryotic cell (e.g. 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 Bio tl which forms are encompassed by the present invention.

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

The expression product is Bio tl having an amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5 or SEQ LD NO:6 or is a derivative, homolog, analog, chemical equivalent or mimetic thereof as defined above or is a derivative, homolog or mimetic having an amino acid sequence of at least about 55% similarity to at least 10 contiguous amino acids in the amino acid sequence as set forth in SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or a derivative or homolog or mimetic thereof.

Another aspect of the present invention is directed to an isolated protein selected from the list consisting of:- (i) protein allergen homolog of Bio tl from a mite or a derivative, homolog, analog, chemical equivalent or mimetic thereof;

(iii) a protein having an amino acid sequence substantially as set forth in SEQ ID NO:2 or SEQ ID NO:4 or one of SEQ ID NO:5 or SEQ ID NO:6 or a derivative, homolog or mimetic thereof or a sequence having at least about 55%> similarity to at least 10 contiguous amino acids in SEQ ID NO:4 or SEQ ID NO: 5 or SEQ ID NO: 6 or a derivative, homolog, analog, chemical equivalent or mimetic of said protein;

(iv) a protein encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:2 or a derivative or homolog thereof or a sequence encoding an amino acid sequence having at least about 55 > similarity to at least 10 contiguous amino acids in SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or a derivative, homolog, analog, chemical equivalent of said protein;

(v) a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ED NO:l or SEQ ID NO:2 or a derivative or homolog thereof or a complementary form thereof under low stringency conditions and which encodes an amino acid sequence substantially as set forth in SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or a derivative or homolog or mimetic thereof or an amino acid sequence having at least about 55% similarity to at least 10 contiguous amino acids in SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6;

(vi) a protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or (v) in a homodimeric form; and (vii) a protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or (v) in a heterodimeric form.

The Bio tl of the present invention may be in multimeric form meaning that two or more molecules are associated together. Where the same Bio tl molecules are associated together, the complex is a homomultimer. An example of a homomultimer is a homodimer. Where at least one Bio tl is associated with at least one non-Bio tl 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 Bio tl permits the large scale production of Bio tl for commercial use. The Bio tl 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 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 Bio tl .

Bio tl 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 CM- cellulose ion exchange chromotography followed by Sephadex (e.g. G-50 column) filtration. Many other techniques are available including HPLC, PAGE amongst others. Once purified, the Bio tl molecule can be partially sequenced and/or fragments produced induced directly as a source of Bio tl or at a template for amino acid synthesis. Blo tl may be synthesized by solid phase synthesis using F-moc chemistry. Bio tl 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 Bio tl 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 Bio tl 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 Bio tl hypersensitivity condition in a subject said method comprising administering to said subject an effective amount of Bio tl or a derivative, homolog, analog, 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 Bio tl molecule such as an epitopic region of Bio tl. The Bio tl 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 (e.g. humans, monkeys), livestock animals (e.g. sheep, cows, horses, donkeys, goats, pigs), laboratory tests animals (e.g. rats, guinea pigs, rabbits, hamsters), companion animals (e.g. dogs, cats), captive wild animals (e.g. emus, kangaroos, deer, foxes) avies (e.g. chickens, ducks, bantoms, pheasants, emus, ostriches), reptiles (e.g. lizards, snakes, frogs) and fish (e.g. trout, salmon).

Reference to "Bio tl hypersensitivity condition" should be understood as a reference to any of type I, II, III or IN hypersensitivity conditions directed to all or part of Bio tl. More particularly, the Bio tl 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 Bio tl, it may be desirable to induce or up regulate 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 lo tl region, it would be desirable to up regulate an immune response to Bio tl .

Accordingly, another aspect of the present invention relates to a method of modulating, in a subject, an immune response directed to Bio tl said method comprising administering to said subject an effective amount of Bio tl or a derivative, homolog, analog, 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 Bio tl or derivative, homolog, analog, 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 Bio tl portion, very low or very high concentrations of Bio tl may be administered to induce low or high dose tolerance, respectively. Alternatively, where it is sought to induce an immune response, doses of Bio tl which do not induce tolerance may be administered. The present invention further extends to the use of Bio tl 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 Bio tl as hereinbefore defined.

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

hi accordance with these methods, more than one type of protein or peptide may be administered. For example, where the Bio tl is administered for the purpose of inducing tolerance, the Bio tl may be co-administered with other known tolerance inducing compounds or molecules. Alternatively, where the Bio tl is administered to up regulate the immune response, the Bio tl 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 days between the administration of the two or more types of molecules. The Bio tl and other compound or molecule may be 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 Bio tl 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 Bio tl and another compound or molecule with which it is co-administered. 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 enumerated above, as required, followed by, for example, filter sterilization or sterilization by other appropriate 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. Narious 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 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 (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved and (b) 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. Alternative amounts include for about 0.1 ng/kg body weight about 100 mg/kg body weight or from 1.0 ng/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 Bio tl or derivative, homolog 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, homolog or mimetic or said protein allergen.

Preferably, the nucleotide sequence encodes the amino acid sequence substantially as set forth in SEQ ID NO:2 or one of SEQ ID NO:5 or SEQ ID NO:6 or a derivative, homolog or mimetic thereof or having at least about 55% or greater similarity to at least 10 contiguous amino acids in SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6. Even more preferably, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:l or SEQ ID NO:2, or a derivative, homolog or mimetic thereof, or capable of hybridizing to SEQ ID NO:l or SEQ ID NO:2 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 Bio tl or a derivative or homolog 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, RSN promoter and SN40 promoter and is preferably pCMN. 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.

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.

Yet another aspect of the present invention is directed to antibodies to Bio tlor Bio tl and their derivatives, homologs, analogs, 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 Bio tl immunoassays or used as antagonists to inhibit Bio tl activity under certain circumstances such as where temporary hypersensitivity inhibition only is required. Techniques for such immunoassays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of Bio tl levels may be important for monitoring certain therapeutic protocols.

Antibodies to the Bio tl (or its derivatives, homologs, analogs 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 Bio tl. The latter would be important, for example, as a means for screening for levels of Bio tl in a biological fluid or purifying Bio tl 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 anti- immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of Bio tl.

Both polyclonal and monoclonal antibodies are obtainable by immunization with Bio tl 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 Bio tl 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 preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes 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 Bio tl. Accordingly, another aspect of the present invention contemplates a method for detecting antibody directed to all or part of Bio tl in a biological sample from a subject said method comprising contacting said biological sample with Bio tl or a derivative, homolog, analog, 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 Bio tl (for example, in a dust sample) or antibody to Bio tl 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 labeled antibody to a target, such as Bio tl.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention to detect Bio tl or antibody to Bio tl. 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 unlabeled antibody of Bio tl 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 Bio tl -antibody) complex, a second antibody specific to the complex molecules, labeled 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-labeled antibody. Any unreacted material is washed 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 labeled 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 Bio tl 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 Bio tl 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 (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. 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.

An alternative method involves immobilizing the target molecules (such as Bio tl) and then exposing the immobilized target to a sample which is to be tested for the presence of antibody to Bio tl. A second labeled 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 (i.e. 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, β-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-labeled 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 antibody- antigen-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.

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-labeled 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 labeled antibody is allowed to bind to the first antibody- hapten 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 non- limiting Examples.

EXAMPLE 1 Mite culture

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 (Bronswijk et al, Acarologia 15: 477-489, 1997) and isolated under a stereomicroscope from the dust samples, which were used for setting-up a starter culture. Fine powdered tetramin fish feed was used as a culture medium for culturing Bt. The tetramin flakes were heated at 60°C for 3 hours before being grounded to kill pre-existing mites and insects. It was then sieved tlirough a 125 μm sieve before use. In order to provide a maximum surface area, a thin layer of <125 μm 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°C and a mean RH of 80%. Figure 1 shows a culture apparatus for dust and storage mites.

EXAMPLE 2

5 '-Rapid Amplification ofcDNA Ends (5 '-RACE) and 3 '-Rapid Amplification ofcDNA Ends (3 '-RACE)

The 5'end of Bio tl cDNA clone was isolated using the 5'-Rapid Amplification of cDNA Ends (5 '-RACE) following the protocol described in the SMART (trade mark) 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 μg of total RNA isolated from B. tropicalis culture. 5'- RACE Ready cDNA reaction mixture composed of 1 μl total RNA sample (1 μg), 1 μl 5'- cDNA Synthesis (CDS) primer, 1 μl SMART II oligo and 2 μl RNAse-free water. 3'- RACE Ready cDNA reaction mixture composed of 1 μl of total RNA sample (1 μl), 1 μl 3 '-CDS primer and 3 μl of RNAse free water. These two reaction mixtures were incubated at 70°C for 2 minutes followed by cooling on ice for 2 minutes. For the 5 μl 5' RACE reaction mixture was then mixed with 2 μl 5X first-strand buffer, 1 μl 20 mM DTT, 1 μl 10 mM dNTP mix and 1 μl Superscript II (200 units/μl; Gibco BRL, USA). The reaction mixture was then incubated at 42°C for 90 minutes in an air incubator prior to dilution with 100 μl of Tricine-EDTA Buffer. The diluted reaction product was then heated at 72°C for 7 minutes. 3' RACE reaction mixture was treated in the same way up to this stage.

5'-Rapid Amplification of cDNA Ends (5'-RACE) was performed using a 50 μl PCR reaction mixture composed of 2.5 μl 5 '-RACE-Ready cDNA, 5 μl of 10X Universal

Primer Mix (UPM), 1 μl of 10 μM Gene Specific Primer 1 (GSP1) and 41.5 μl of Master

Mix (34.5 μl PCR grade water, 5 μl 10X Advantage 2 PCR Buffer, 1 μl 10 mM dNTP Mix and 1 μl 50X Advantage 2 Polymerase Mix). Another 50 μl PCR reaction mixture composed of 2.5 μl 5 '-RACE-Ready cDNA, 5 μl of 10X Universal Primer Mix (UPM), 1 μl of 10 μM Gene Specific Primer 2 (GSP2) and 41.5 μl of Master Mix (34.5 μl PCR grade water, 5 μl 10X Advantage 2 PCR Buffer, 1 μl 10 mM dNTP Mix, and 1 μl 50X

Advantage 2 Polymerase Mix) was set up simultaneously. Positive and negative controls were prepared according to protocol. Gene-Specific Primer 1 (GSP1), 5' CC GCG TGG

AAT TGT ATC ACC ATG GC 3' [SEQ ID NO:13], and Gene-Specific Primer 2 (GSP2), 5' CC GTA ACC AAC AAT GTT GAC GGC 3' [SEQ ID NO: 14] were designed based on the nucleotide sequences of Group 1 allergens of other mite species, with a T_m of 66°C and

60°C, respectively.

3'-Rapid Amplification of cDNA Ends (3'-RACE) was performed using a 50 μl PCR reaction mixture composed of 2.5 μl 3 '-RACE-Ready cDNA, 5 μl of 10X Universal Primer Mix (UPM), 1 μl of 10 μM Gene Specific Primer 3 (GSP3) and 41.5 μl of Master Mix (34.5 μl PCR grade water, 5 μl 10X Advantage 2 PCR Buffer, 1 μl 10 mM dNTP Mix and 1 μl 50X Advantage 2 Polymerase Mix). Another 50 μl PCR reaction mixture composed of 2.5 μl 3 '-RACE-Ready cDNA, 5 μl of 10X Universal Primer Mix (UPM), 1 μl of 10 μM Gene Specific Primer 4 (GSP4) and 41.5 μl of Master Mix (34.5 μl PCR grade water, 5 μl 10X Advantage 2 PCR Buffer, 1 μl 10 mM dNTP Mix and 1 μl 50X Advantage 2 Polymerase Mix) was set up simultaneously. Positive and negative controls were prepared according to protocol. Gene-Specific Primer 3 (GSP3), 5' GGT GCC ATC AAC CAT TTG TCC G 3' [SEQ ID NO:15] and Gene-Specific Primer 4 (GSP4), 5' CCA ATC CGT ATG CAA GGA GGC 3' [SEQ ID NO: 16] were designed also based on the nucleotide sequences of Group 1 allergens of other mite species with a T_m of 61°C and 58°C, respectively.

5' and 3 '-Rapid Amplification of cDNA Ends (5 'RACE and 3' RACE) were performed by 30 cycles using the PTC- 100 (trade mark) Programmable Thermal Controller (MJ Research Inc, USA). Each cycle consists of 94°C for 5 seconds, 55°C for 10 seconds and 72°C for 3 minutes.

5' RACE and 3' RACE products were initially analyzed by agarose gel electrophoresis using 1% w/v agarose/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. Purified 5 'RACE and 3' RACE products were finally analyzed by DNA sequencing using the ABI 377 DNA Sequencer (Applied Biosystems).

One clone was isolated from the 5 'RACE product. It was a truncated gene lacking the 3' region. This clone was designated as clone Bio tlR2.

EXAMPLE 3 TOPO TA cloning of the clone Bio tlR2

Bio tlR2 was ligated into pCR2.1-TOPO cloning vector and was subsequently transformed into TOPOlO One Shot (trade mark) competent cells using the TOPO (trade mark) TA

Cloning version E (Invitogen). In order to do this, a single 3'-A overhang was introduced to the clone by incubating in a 25 μl PCR reaction consisting 1 μl (5 U/μl) of Taq Polymerase (Promega, USA), mixed well and incubated at 72°C for 8 to 10 minutes. It is not necessary to change buffer. 4 PCR reactions were set up for this purpose.

The reaction was then purified using the GFX (trade mark) PCR DNA and Gel Band Purification Kit (Pharmacia). The 100 μl reaction mix was added to 500 μl 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 μl 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 μl 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 μl of the purified Bio tlR2 with 3'-A overhang added to 2 μl of deionized water and 1 μl of pCR2.1-TOPO vector. The ligation reaction was incubated for 5 minutes at room temperature. Transformation was then performed by mixing 2 μl of the TOPO (trade mark) Cloning reaction into a vial of TOP 10 One Shot (trade mark) competent cells which was previously treated with 2 μl of 0.5 M β-mercaptoethanol. After incubating the One Shot (trade mark) transformation reaction for 30 minutes on ice, the cells were heat-shocked at 42°C for 30 seconds in a water bath followed by 2-minute incubation on ice. 250 μl 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°C. Transformed colonies and white colonies, were subcultured overnight at 37°C on LB medium with ampicillin. Plasmid DNA purified by Wizard Plus SN minipreps DΝA purification system (Promega) were analyzed by DΝA sequencing using the ABI 377 DΝA Sequencer (Applied Biosystems). EXAMPLE 4 Full length isolation of Bio tl

m order to generate the full length of Bio tl, specific primers were again designed base on the known sequence of Group 1 allergens and Bio tlR2. 5'-Rapid Amplification of cDNA Ends (3 '-RACE) was performed using a 50 μl PCR reaction mixture composed of 2.5 μl 5 '-RACE-Ready cDNA, 5 μl of 10X Universal Primer Mix (UPM), 1 μl of 10 μM Gene Specific Primer 5 (GSP5) and 41.5 μl of Master Mix (34.5 μl PCR grade water, 5 μl 10X Advantage 2 PCR Buffer, 1 μl 10 mM dNTP Mix, and 1 μl 50X Advantage 2 Polymerase Mix). Another 50 μl PCR reaction mixture composed of 2.5 μl 5 '-RACE-Ready cDNA, 5 μl of 10X Universal Primer Mix (UPM), 1 μl of 10 μM Gene Specific Primer 6 (GSP6) and 41.5 μl of Master Mix (34.5 μl PCR grade water, 5 μl 10X Advantage 2 PCR Buffer, 1 μl 10 mM dNTP Mix and 1 μl 50X Advantage 2 Polymerase Mix) was set up simultaneously. Positive and negative controls were prepared according to protocol. Gene- Specific Primer 5 (GSP5), 5' CCA ACT GTT TCG TAC GAT CCA ATA ATC 3' [SEQ ID NO: 17], and Gene-Specific Primer 6 (GSP6), 5' ACA ACC GTG TTG GGA AGC ACA ATC GAC TAA TTC TTG 3' [SEQ ID NO:18] were designed also bases on the nucleotide sequences of clone Bio tlR2 and Group 1 allergens of other mite species with a T_m of 58°C and 71°C, respectively. 5' RACE product using the specific primer 5(GSP 5) produced a positive product Bio tlR3 and it was confirmed by sequencing that this was the 3' end of the gene Bio tl. This portion of the gene has common overlapping sequences with the clone Bio tlR2. The full gene was thus identified.

EXAMPLE 5 Hybridization of 5' RACE and 3 '-RACE product to generate full length recombinant Bio tl gene

The full-length Bio tl cDNA gene was generated by performing polymerase chain reaction using 5 '-RACE and 3 '-RACE products as the DNA templates. Based on the known nucleotide sequences from these two portions of Bio tl gene, i.e. Bio tlR2 and Bio tlR3, specific primers (forward and reverse primers) were designed. The primers were designed so that the full length of Bio tl gene could be generated in the PCR reaction.

The above procedure was carried out so as to determine the presence of the polymorphic forms of Bio tl. Hybridization of Bio tlR2 and Bio tlR3 could generate a single full length gene only.

PCR was performed by using 60 ng each of the 5' RACE product and 3 '-RACE product DNAs. A PCR reaction mixture of 25 μl was prepared using the forward specific primer, 5' TGT GGA ACG CAA ACC CAA ATC CAA AAA ACG 3' [SEQ ID NO: 19] and the reverse specific primer, 5' AGG GCA AGC AGT GGT AAC AAC GCA GAG TAC 3' [SEQ ID NO:20]. The DNA strands were denatured at 94°C for 30 seconds followed by annealing at 72°C for 5 minutes. Amplification was performed by 30 cycles using the PTC- 100 (trade mark) Programmable Thermal Controller (MJ Research Inc., USA). Each cycle consists of 94°C for 30 seconds, 55°C for 30 seconds and 72°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).

EXAMPLE 6

TOPO TA cloning of full-length Bio tl cDNA gene

The full-length Bio tl cDNA gene was ligated into pCR2.1-TOPO cloning vector and was subsequently transformed into TOP 10 One Shot (trade mark) competent cells using the TOPO (trade marm) TA Cloning version E (Invitrogen). The procedure is the same as mentioned above in Example 3. EXAMPLE 7 Sub-cloning of Bio tl insert into GST-fusion expression vector in E. coli

The Bio tl cDNA insert was released and ligated to pGEX-4Tl vector with EcoRI and Notl restriction enzyme site and transformed to DH5α. The fusion protein was produced by 1 mM IPTG induction and purified using glutathione Sepharose. Expression in E. coli is shown in Figure 6A.

EXAMPLE 8 Sub-cloning of Bio tl insert intopPIC9 expression vector in yeast

The Bio tl cDΝA insert was released and ligated to PIC9 vector with Xhol and Notl restriction enzyme sites, and transformed to DH5α. The plasimd was then isolated and linearized with BgHl and then transformed into the yeast GS115 for protein expression. GS115 has a defect in the histidinol dehydrogenase gene(His4) which prevents it from synthesizing histidine. All expression plasmids carrying the wild-type His4 gene which complement His4 in the host, so fransformants are selected for their ability to grow on histidine-deficient medium. Transformants of GS115 with BglR 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 are then grown overnight in 1 ml of BMGY medium (1% v/v yeast extract, 2% v/v peptone, 100 mM potassium phosphate, pH 6.0, 1.34%> v/v yeast nitrogen base with ammonium sulfate, 0.00004%) v/v biotin, 1%> v/v glycerol). Overnight cultures were then transferred to a larger volumn and grow to O>D 2-6 at 600 nm wavelength to log phase. Then the cells are spun down and resuspended in BMMY medium (0.5% v/v methanol replaces the glycerol of BMGY medium) with its volume reduced to 1/10 of its earlier volume in BMGY. The culture is allowed to grow for 2 to 3 days in the presence of methanol (final concentration of 0.5% to the total volumn). The methanol is added daily and the sample is drawn for the recombinant protein expression for analysis on an SDS- PAGE (10%) w/v). Expression in yeast is shown in Figure 6B.

EXAMPLE 9 DNA sequence analysis

The complete sequence consists of 1108 base pair (Figures 2 and 3), including 38 base pair long 5' non- translating region. The sequence has an ATG start codon at position 40 to 42 and a stop codon, TAA at position 1039 to 1041. This shows an open reading frame coding for a 330 amino acids. The predicted molecular weight of the matured protein is approximately 39 kilodaltons.

Sequence alignment using the CLUSTALW program indicates that the Bio tl has a 53% homology with Der pi from Dermatophagoides pteronyssinus, 50 % homology with Der fl from Dermatophagoides farinae and 51%> homology with Eur ml from Euroglyphus maynei. The homology among group 1 allergens of Dermatophagoides pteronyssinus, Dermatophagoides farinae and Euroglyphus maynei is greater than 80%> (see Figure 4).

EXAMPLE 10 Protein sequence analysis

Protein sequence homology search was performed by using the Advance BLAST search from NCBI. Bio tl has a 34% homology with Der pi from Dermatophagoides pteronyssinus, 31% homology with Der fl from Dermatophagoides farinae and 36% homology with Eur ml from Euroglyphus maynei. The homology among group 1 allergens of Dermatophagoides pteronyssinus, Dermatophagoides farinae and Euroglyphus maynei is greater than 82%.

As compared to group 1 allergens of other species of mites, the putative amino acid sequence of Bio tl (Figure 5) has two regions A and B - distinctly different. The pi values for Eur ml, Der pi, Der fl and Bio tl are 5.93, 5.63, 5.76 and 8.44, respectively. The molecular weights for these allergens are 25.1, 25, 25.2 and 39 kilodalton, respectively.

The expression in E. coli is low and the molecular weight of the fusion protein GST-Bio tl is about 70 kD whereas the protein expressed in yeast GS115 is high and the molecular weight is about 39 kD (Figure 5).

EXAMPLE 11 Production of Bio tl monoclonal antibodies, isolation of native Bio tl and its IgE reactivity

DNA/Protein Immunization

Five female, 5-6 week old BalbC/J mice were immunized with 50 μg pSecTag-Blo tl plasmid DNA resuspended in 50 μl of PBS. Plasmids were administered intramuscularly (πvl) on the quadriceps muscle of the hindleg of each mice using the Terumo U-100 Insulin syringe with needle. Each immunized mice were pulsed on the site of the immunization using the Electro Square Porator (ECM 830) attached to a 2-needle array electrode with cable (Model No. 532) and 2-needle array (Model No. 533) (BTX, Genetronix, Inc., CA, USA). The following parameters were used: Voltage: 82, Pulse length: 20 msec, Pulse: 4, Interval: 200 msec. Each mouse received 2 booster doses at days 14 and 28.

To produce high titer of antibodies, the 5 DNA-immunized mice were further boosted with recombinant protein expressed in yeast (yBlo tl) at days 42, 56 and 70 by IP (Intraperitoneal) route. Fifty μg of yBlo tl in 200 mg alum suspended in 200 μl PBS was administered intra-peritoneally on each mouse using a 27.5 G needle.

Blood was extracted infra-orbitaly every week to monitor the titer of antibody production. Blood sera were stored at -180°C until used. Enzyme-linked immunosorbent assay

The titer of antibody production for each DNA immunized mouse was determined by enzyme linked immusorbent assay (ELISA). Recombinant Bio tl (5 μg/well) and Bio t crude extract (100 μg/well) was coated in duplicates onto ELISA plates overnight at 4°C using 50 μl of 0.1M NaHCO₃. Plates were blocked with 1% v/v BSA in PBST (0.05% w/v Tween 20) for 1 hour at room temperature. ELISA plates were then incubated for 1 hour each at room temperature with serially diluted mouse sera followed by anti-mouse Ig biotin-conjugated antibody diluted 5000X (Sigma) and finally with 5000X dilution of ExtrAvidin (Sigma). After thorough washing steps with PBST between incubations, colorimetric reaction was performed using β-nitrophenyl phosphate (PNPP) tablets (Sigma). Optical density (OD) index was determined by reading the absorbance at 450 nm using the Spectra & Rainbow ELISA reader.

Culture of parental myeloma cells (X63A8.653)

A week prior to fusion, parental myeloma cells were cultured and grown to confluence. In brief, a vial of X63Ag8.653 myeloma cells from the liquid nitrogen tank was thawed at 37°C in a water bath. Cells were gently pipeted into a conical 15 ml centrifuge tube containing 10 ml of Medium A which has been pre-warmed to 37°C. Cell suspension was then centrifuged gently at 400 g (1000 rpm using the Sorvall RT7) for 5 minutes and the supernatant discarded. Cells were resuspended with 1 ml warm Medium A-ClonaCell-HY (StemCell Technologies Inc.) and were transfered into 50 ml tissue culture flask containing 50 ml Medium A. Cells were cultured to confluence for 2 to 3 days with 5% v/v CO₂ at 37°C. Cells were subcultured into several flasks reaching the mid log phase of growth on the day of fusion.

Fusion and Cloning of Hybridoma Clones

Fusion of myeloma and spleen cells and cloning of hybridoma clones were done using the ClonaCell-HY Hybridoma Cloning Kit (StemCell Technologies Inc.). In brief, 2x10⁷ viable myeloma cells were counted and resuspended in 30 ml of Medium A. Simultaneously, 2 mice with highest titer of antibody production were sacrificed by cervical dislocation and the spleens dissected under sterile conditions. Splenocytes were separated by grinding the spleen tissue using the course side of a glass slide and were resuspended in Medium B. After counting cells using the Tryphan blue exclusion assay, 1x10 viable cells were fused with previously prepared mayeloma cells using PEG solution and Medium B. Hybridomas were then incubated overnight at 37°C in Medium C. Hybridoma clones were then plated using medium D (methylcellulose-based medium) and were incubated for 14 days at 37°C until colonies appeared for picking. Isolated colonies were picked using a pipet tip and were cultured in 200 μl Medium E using a 96-well tissue culture plates. Clones were finally screened for antibody production by ELISA.

Screening and Isotyping of Hybridoma Clones

Hybridoma clone supernatants were collected and were used for detection of antibody production against recombinant Bio tl and crude Bio t extract by ELISA following the protocol mentioned above. Medium E alone was used as control in all ELISA protocols. Antibody-producing clones against Bio tl and/or crude Bio t extract were subcultured and expanded into 6-well plates using complete DME medium (Hyclone) with 10% w/v Fetal Calf Serum (Hyclone). Isotyping of each positive clone was done by ELISA using biotin- conjugated anti-mouse IgGl, IgG2a, IgG2b, IgG3, IgM, IgA, IgE, and IgD (Pharmingen). High antibody-titer producing clones were expanded and frozen for ascites production.

Ascites Production

High-titer monoclonal anti-Bio t 1 antibodies were obtained from ascitic fluid of Balb/c mice inoculated with Bio tl hybridoma cells. One week prior to inoculation of hybridoma cells, each mouse were injected intraperitoneally with 1 ml Pristane (Sigma) using 22-G needle. Bio tl hybridoma clones, cultured in complete DMEM (Hyclone) medium at log phase-growth, were harvested, washed 3X in PBS, and assayed for viability using the Tryphan Blue exclusion method. 5 X 10⁶ viable cells in 1 ml PBS was injected intraperitoneally into each mouse using 22-G needle. Ascitic fluid was harvested 1 to 2 weeks after cell inoculation using 18-G needle inserted 1-2 cm into the abdominal cavity. Ascites were collectedby gravity flow into 15 ml conical centrifuge tubes. Second ascitic fluid collection was performed 2 to 3 days. After the second collection, mice were sacrificed by cervical dislocation. Collected ascites from different mice injected with the same hybridoma clone were pooled followed by centrifugation at 4200 rpm (Jouan Centrifuge), room temperature. Supernatant was collected, assayed for antibody titer and specificity by ELISA and Western blot. Ascites were stored at -80°C in aliquots until use.

Antibody Purification

Bio t 1 monoclonal antibodies were purified from ascitic fluid by affinity chromatography using Protein G-agarose column (KPL). Previously packed 1 ml Protein G column was re- equilibrated with 10 column- volume (CN) of wash binding buffer (0.1 M ΝaPO4, 0.15 M NaCl, pH 7.4). One ml of ascitic fluid was gently applied onto the column followed by thorough wash with wash/binding buffer until the eluate absorbance at 280 nm approximates 0. Bound antibodies were eluted with 4 ml of elution buffer (0.2 M Glycine, pH 3.0). Fractions were collected at 0.5 ml per tube containing 120 ul of 5X wash/binding buffer to equilibrate the pH of the eluate. Ten μl of each fraction was analyzed under denaturing conditions by SDS-PAGE using 12% w/v Tris-Glycine gel. Antibody- containing fractions were pooled, dialyzed against 0.1 M NaHCO3, 0.5 M NaCl (pH 8.3), and quantified using the DC Protein Assay (BioRad).

Coupling of Purified Antibodies to Sepharose Beads

CNBr-activated sepharose beads (Pharmacia Biotech) were allowed to swell by re- suspending 0.7 g of the beads in 5 ml of 1 mM HC1 for 3-5 minutes. Swollen beads were washed with 200 ml of 1 mM HC1 followed by 10 ml coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3) using a column. Beads were resuspended in 5 ml coupling buffer and added into 1 ml of purified antibody in a 15 ml conical centrifuge tube. The suspension was incubated for 16 to 20 hours at 4°C with constant shaking. Blocking buffer was discarded after centrifugation and the beads were incubated with 4 ml blocking buffer (1 M ethanolamine, pH 8.0) for 2 hours at room temperature. The suspension was finally centrifuged at 4200 rpm (Jouan) and the beads were resuspended in 10 ml IX TBS (pH 7.5). Antibody bound to sepharose beads were analyzed by SDS-PAGE using 12%) w/v Tris glycine denaturing gel.

Preparation of Crude Bio t Protein Extract

Bt mites were cultured in powdered TetraMin fish flakes (Tetra Werke, Germany) for three to four weeks under natural environmental conditions with a mean annual temperature of

30°C and a mean relative humidity of greater then 80%. The mites were harvested using a modified Tullgren apparatus and stored at -80°C until use. Ten grams of frozen mites, wrapped in aluminum foil was soaked in liquid nitrogen for 2 minutes followed by grinding using a precooled mortar and pestle for 2 hours. Extraction was performed using a total volume of 50 ml of IX TBS with 2 mM PMSF and 1 mM EDTA added slowly while grinding the mites. The mite extract suspension was incubated for 16-20 hours at 4°C with constant shaking. After centrifugation of the suspension at 17,000 rpm using the Sorvall

(registered trade mark) Ultra Pro 80 centrifuge, the supernatant was collected and quantified by DC Protein Assay (BioRad). Crude Bio t protein extracts were stored in aliquots at -80°C until use.

Isolation of Native Bio 1 1

Antibody coupled to Sepharose beads were packed onto 10 ml polypropylene column (Pierce) using IX TBS (pH 7.5). The column was washed thoroughly with IX TBS until the OD at 280 nm was 0. Five hundred mg of crude Bio t protein extract (approximately 50 ml extract) was gently passed onto the column followed by thorough wash with IX TBS until the OD at 280 nm was 0. Bound native Bio tl was eluted using 10 ml of elution buffer (0.1 M Glycine, 0.15 M NaCl, pH 2.6). Fractions were collected at 1 ml per tube. Six hundred μl of each fraction was TCA precipitated and resuspended in 20 μl SDS- PAGE sample buffer. Samples were analyzed by denaturing SDS-PAGE using 12%> w/v Tris-Glycine gel followed by silver staining and/or Western blot analysis.

SDS-PAGE

Purified protein samples were analyzed by SDS-PAGE according to the method described in Laemmeli, Nature 227: 680, 1970. hi brief, protein samples were mixed 1:1 with 2X sample buffer and were boiled for 10 minutes. Samples were separated on a 12 % w/v Tris- Glycine denaturing gel using the Mini PROTEAN electrophoresis system (BioRad, USA). Gel was run at constant 110 voltage for 90 minutes. Broad range marker (BioRad) was used as standard.

Silver Staining

SDS-PAGE gel was fixed in 100 ml 50% v/v methanol, 5%> v/v acetic acid in water for 30 minutes at RT with constant shaking. After thorough wash with 100 ml of 50%o v/v methanol in water for 10 minutes and 200 ml water for 60 minutes at RT, the gel was sensitized with 100 ml of 0.02% w/v sodium thiosulphate for 2 minutes at RT with constant shaking. The gel was incubated with 50 ml chilled 0.1% w/v silver nitrate solution for 40 minutes at 4°C with constant shaking after 2 washes with 200 ml water. Staining was developed with 0.04% v/v formalin (37%> v/v formaldehyde in water) in 2%» w/v sodium carbonate with intensive shaking after 2 washes with 200 ml water. The reaction was stopped by incubating the gel with 100 ml stop solution (3.65 g/250 ml EDTA-Na₂- H₂O) followed by 3X wash with 200 ml water. Stained gel was framed and dried using the GelAir Drying Frame Assembly procedure (BioRad, USA).

Western Blot Analysis

The specificity of the Bio tl monoclonal antibodies were analyzed by Western blot analysis, h brief, recombinant Bio tl, crude Bio t protein extract, spent mite medium extract and control protein (GST) were electrophoresed on 12% w/v Tris Glycine gel as described above and were electroblotted onto nitrocellulose membrane (Hybond C, Amersham Life Sciences, USA) using the MiniProtean II cell (BIORAD, USA) at 110 N for 1 hour. Membrane was blocked with 5% w/v skimmed milk in PBS-T (0.05%> w/v Tween 20). After overnight incubation of membrane with 1000X dilution of Bio tl monoclonal antibody at 4°C with constant shaking followed by 6X wash with PBS-T, the membrane was incubated with 5,000X dilution of Biotinylated anti-mouse Ig (Pharmingen). Prior to detection using the SuperSignal (registered trade mark) WestPico Chemiluminescent Substrate (Pierce, 111., USA) for 5 minutes, the membrane was incubated with 5000X dilution of alkaline phosphatase and peroxidase conjugated ExtrAvidin (Sigma) followed by 6X wash with PBS-T. Results were detected by autoradiography using the Kodak Biomax ML film. To confirm the results obtained, the membrane was likewise exposed to the AP Buffer System (BioRad) for color reaction. Detection was performed for 20-30 minutes with IX AP Buffer containing 100 μl each of AP color reagents A and B (BioRad).

Human IgE ELISA

Enzyme linked immunosorbent assay (ELISA) was used to evaluate the profile of sensitization of a panel of asthmatic patients' sera to rocombinant and native Bio tl. The results are shown in Figures 16 to 18. Briefly, 5 μg each of the recombinant and native Bio tl and 100 μg of Bio t crude extract were coated onto ELISA plates overnight at 4°C using 50 μl of 0.1 M ΝaHCO₃. Plates were blocked with 1% w/v BSA (Sigma) in PBST for 1 hour at room temperature. ELISA plates were incubated for 1 hour each at room temperature with human sera diluted 5X in blocking buffer followed by 1-hour incubation with biotinylated anti-human IgE (Pharmingen, CA, USA) diluted lOOOx in blocking buffer. Plates were incubated with 2000X dilution of ExtrAvidin- AP conjugate (Sigma) for 1 hour. Finally, colorimetric reaction was performed using p-nitrophenyl phosphate (Sigma). Absorbance at 405 nm was determined using the Spectra & Rainbow ELISA reader (Tecan, Austria). Detection of Bio tl antibodies

High titer of Bio tl antibodies were detected among the 5 immunized Balb/c mice following the immunization schedule outlined in Figure 7. Total Ig titers were detected from the blood extracted 7 days after each boost followed by ELISA using recombinant Bio tl expressed in E. coli and Pichia pastoris. Figures 8 and 9 shows the kinetics of antibody production of each mouse immunized with Bio tl.

Splenocytes from 2 mice (Ml and M3) with the highest titer of antibody production were successfully fused with cultured X63 myeloma cells as evident in the number of clones observed on methylcellulose plates. 1,632 clones were picked using a sterile 200 μl pipet tip and out of these, 1,495 grew and were screened antibody production by ELISA. Primary screening resulted in 237 clones with positive total Ig production against the recombinant yBlo tl and/or crude Bio t protein extract. 45 clones with high titers of anti Bio tl were expanded and screened for Ig isotypes by ELISA. 6 of these clones (Table 3) were selected for ascites production. Figure 10 shows the total Ig titer production of the 6 hybridoma clones used for ascites production. Antibody titers were assayed by ELISA using the culture supernanats.

TABLE 3 Isotypes of six Bio tl hybridoma clones used for ascites production

Western blot analysis

The results of Western blot analysis are shown in Figure 11. The results illustrate the specificity of Bio tl monoclonal antibody 11F1. The monoclonal antibody was able to recognize both the recombinant Bio tl expressed in E. coli and Pichia pastoris expression systems as well as single distinct bands in both the crude Bio t protein extract and spent- mite medium extract but not in the GST control.

Figure 12 shows the purification of the Bio tl monoclonal antibody 11F1 using Protein G column. Contaminating proteins present in the ascitic fluid extracted from the immunized mice were successfully isolated from the antibody as shown by the absence of other bands aside from the heavy and light chain on the purified antibody fraction (last lane).

Figure 13 shows the silver stained SDS-PAGΕ gel of the recombinant and native Bio tl isolated using the sepharose-coupled Bio tl monoclonal antibody 11F1. A single distinct band between the 45 and 31 kD markers was observed in all fractions during the affinity chromatography purification.

Table 4 summarizes the IgΕ reactivity of the purified-native Blot 1 in comparison with the recombinant E. coli and yeast Bio tl as determined by ΕLISA. 63 Singaporean (Table 5) and 70 Malaysian (Table 6) Bio t extract positive sera were used in the experiment. Similar frequency of IgΕ reactivity was observed between yBlo tl and native Bio tl with the Singaporean sera (both at 63%). On the other hand, a higher frequency of IgΕ reactivity was observed with the yBlo tl in comparison with the native Bio tl among the Malaysian sera. However, if the titer of human IgΕ is plotted against yeast expressed Bio tl (yBlo tl), GST-Bio tl and native Bio tl (nBlo tl), it is clearly seen that the IgΕ titers of the native Bio tl are much higher in both Singapore as well as Malaysian panels of sera as compared to those of yBlo tl or GST-Bio tl (Figures 13, 14 and 15). T ABLE 4 Summary of IgE ELISA

TABLE 5 IgE data with 63 Singaporean sera reacting with yBlo tl, GST-Blo tl and nBlo tl

TABLE 6 IgE reactivity of 70 Malaysian sera with bBlo tl , GST-Blo tl and nBlo tl

Figures 16-18 show IgeE reactivity of sera from 40 adult rhinitis (Figure 16), 40 adult asthma (Figure 17) and 40 asthmatic children (Figure 18).

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. BIBLIOGRAPHY

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Claims

1. An isolated nucleic acid molecule or derivative thereof comprising a nucleotide sequence encoding or complementary to a sequence encoding protein allergen Bio tl or its equivalent from a mite or a derivative, homolog or mimetic of said protein allergen.

2. The isolated nucleic acid molecule of Claim 1 wherein the mite is a dust mite or storage mite.

3. The isolated nucleic acid molecule of Claim 1 wherein the dust or storage mite is B. tropicalis.

4. An isolated nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding Bio tl as hereinbefore defined from B. tropicalis.

5. The nucleic acid molecule of Claim 4 comprising a nucleotide sequence encoding, or a nucleotide sequence complementary to a nucleotide sequence encoding, an amino acid sequence substantially as set forth in SEQ ID NO:2 or SEQ ID NO: 4 or SEQ ID NO:5 to SEQ ID NO:6 or a derivative, homolog or mimetic thereof or having at least about 55% or greater similarity to at least 10 contiguous amino acids in SEQ ID NO: 3 or one of SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 -

6. The nucleic acid molecule of Claim 4 comprising a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:2, or a derivative, homolog or mimetic thereof or capable of hybridizing to SEQ ID NO:l or SEQ ID NO:2 under low stringency conditions.

7. The nucleic acid molecule or derivative thereof comprising a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ ID NO:2 or a derivative, homolog or mimetic thereof or a complementary form thereof or capable of hybridizing to SEQ ID NO:l or SEQ ID NO:2 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:4 or SEQ ID NO:5 to SEQ ID NO:6 or a sequence having at least about 55% similarity to at least 10 contiguous amino acids in SEQ ID NO:4 or SEQ LD NO:5 or SEQ LD NO:6 -

8. The nucleic acid molecule of Claim 7 comprising a sequence of nucleotides substantially as set forth in SEQ LD NO:l or SEQ ID NO:2.

9. An isolated nucleic acid molecule comprising a nucleotide sequence encoding or complementary to a sequence encoding Bio tl from B. tropicalis, said nucleotide sequence is set forth in SEQ ID NO:l or SEQ ID NO:2.

10. A genomic nucleic acid molecule or derivative thereof capable of hybridizing to SEQ ID NO:l or SEQ LD NO:2, or a derivative or homolog or mimetic thereof, under low stringency conditions of 42°C.

11. A cDNA sequence comprising a sequence of nucleotides as set forth in SEQ ID NO:l or SEQ ID NO:2 or a derivative thereof including a nucleotide sequence having similarity to SEQ ID NO: 1 or SEQ LD NO:2.

12. An isolated Bio tl protein having an amino acid sequence set forth in SEQ LD NO:2 or SEQ LD NO:4 or one of SEQ LD NO:5 to SEQ LD NO:6 or is a derivative, homolog, analog, chemical equivalent or mimetic thereof having at least about 55% similarity to at least 10 contiguous amino acids in the amino acid sequence as set forth in SEQ LD NO:2 or SEQ ID NO:4 or SEQ LD NO:5 to SEQ LD NO:6 or a derivative or homolog or mimetic thereof.

13. An isolated protein selected from the list consisting of:- (i) protein allergen Bio tl homolog from a mite or a derivative, homolog, analog, chemical equivalent or mimetic thereof;

(iii) a protein having an amino acid sequence substantially as set forth in SEQ ID NO:2 or SEQ ID NO:4 or one of SEQ LD NO:5 to SEQ ID NO: 6 or a derivative, homolog or mimetic thereof or a sequence having at least about 55% similarity to at least 10 contiguous amino acids in SEQ LD NO:4 or SEQ LD NO:5 or SEQ LD NO:6 or a derivative, homolog, analog, chemical equivalent or mimetic of said protein;

(iv) a protein encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:l or SEQ LD NO:2 or a derivative or homolog thereof or a sequence encoding an amino acid sequence having at least about 55 > similarity to at least 10 contiguous amino acids in SEQ ID NO:4 or SEQ LD NO:5 or SEQ LD NO:6 or a derivative, homolog, analog, chemical equivalent of said protein;

(v) a protein encoded by a nucleic acid molecule capable of hybridizing to the nucleotide sequence as set forth in SEQ ID NO:l or SEQ ID NO:2 or a derivative or homolog thereof or a complementary form thereof under low stringency conditions and which encodes an amino acid sequence substantially as set further in SEQ ID NO:2 or SEQ ID NO:4 or one of SEQ LD NO:5 or SEQ LD NO:6 or a derivative or homolog or mimetic thereof or an amino acid sequence having at least about 55% similarity to at least 10 contiguous amino acids in SEQ LD NO:2 or SEQ ED NO:4 or one of SEQ LD NO:5 or SEQ ID NO: 6; (vi) a protein as defined in paragraphs (i) or (ii) or (iii) or (iv) or (v) in a homodimeric form; and

14. 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 Bio tl allergen from said B. tropicalis or a derivative, homolog, analog, mimetic or chemical equivalent thereof for a time and under conditions sufficient to desensitize said individual.

15. A method of modulating, in a subject, an immune response directed to B. tropicalis allergen Bio tl, said method comprising administering to said subject an effective amount of said allergen or a derivative, homolog, analog, chemical equivalent or mimetic thereof for a time and under conditions sufficient to up regulate, down regulate or otherwise modulate said immune response.

16. 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 Bio tl or a derivative or homolog thereof whereby airway hyper-reactivity or airway inflammation is prevented.

17. The method of Claim 16 wherein the allergic condition is allergic asthma, atopic dermatitis and/or rhinitis.

18. The method of Claim 16 or 17 wherein the nucleic acid molecule is in the form of an eukaryotic expression vector.

19. The method of Claim 16 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.

20. The method of Claim 16 wherein the individual is a human.

21. The method of Claim 16 wherein the vector is a plasmid vector.

22. The method of Claim 16 wherein the administration is via intramuscular injection.

23. Use of a B. tropicalis allergen Bio tl or a derivative or homolog thereof in the manufacture of a medicament for modulating an immune response.

24. An agent useful for modulating an immune response, said agent comprising a B. tropicalis allergen Bio tl or a derivative or homolog thereof.

25. A composition for use in modulating an immune response comprising a B. tropicalis allergen Bio tl or a derivative or homolog thereof and one or more pharmaceutically acceptable carriers and/or diluents.

26. Antibodies to B. tropicalis allergen Bio tl and its derivatives, homologs, analogs, mimetics and chemical equivalents thereof.

27. A method for detecting antibody directed to all or part of B. tropicals allergen Bio tl in a biological sample from a subject, said method comprising contacting said biological sample with said allergen or a derivative, homolog, analog, chemical equivalent or mimetic thereof for a time and under conditions sufficient for an antibody- protein complex to form, and then detecting said complex.

28. A pharmaceutical composition useful for modulating an immune response directed to B. tropicalis, said composition comprising Bio tl allergen from B. tropicalis or derivative, homolog, analog, chemical equivalent or mimetic thereof and one or more pharmaceutically acceptable carriers and/or diluents.