CLONING AND SEQUENCING OF ALLERGENS OF DERMATOPHAGOIDES (HOUSE DUST MITE)

Abstract: Isolated DNA encoding a protein allergen or an epitope of a protein allergen of Dermatophagoides farinae or Dermatophagoides pteronyssinus. An isolated protein allergen or peptide which includes an epitope of a protein allergen of Dermatophagoides farinae or Dermatophagoides pteronyssinus. Diagnostic and therapeutic compositions and methods of use thereof.

Description: ΓT.ONTNΠ AMΠ κητiRMπτwG OF AT.T.RWΠRWS OF DRRMAτnPHAr:nτnκς (HOUSE nτιςτ MTTF Descri tion Background Recent reports have documented the importance of responses to the Group I and Group II allergens in house dust mite allergy. For example, it has been documented that over 60% of patients have at least 50% of their anti-mite antibodies directed towards these proteins (Lind, P. et.al.., Allergy, 3S:259-274 (1984); van der Zee, J.S. et.al., .Allergy C\in. Tmmnnnl . , £1:884-896 (1988)). It is possible that children show a greater degree of reactivity (Thompson, P.J. et.al., Tmmnnnlngy, 4:311-314 (1988)) . Allergy to mites of the genus 5£IlB£££Ell£S2i^££ (^-) - s associated with conditions such as asthma, rhinitis and ectopic dermatitis. Two species, D_. , r£ γ inu and D_. .fa.rΪ£ e , predominate and, as a result, considerable effort has been expended in trying to identify the allergens produced by these two species. D^_ ££®££BΣ £iBil£ mites are the most common Dermatojha^ _~___.__f__i species in house dust in Western Europe and Australia. The species D^farinae predominates in other countries, such as, North America and Japan (Wharton, G.W. , J. Medical Entom, 12:577-621 (1976)). It has long been recognized that allergy to mites of this genus is associated with diseases such as asthma, rhinitis and atopic dermatitis. It is still not clear what allergens produced by these mites are responsible for the allergic response and associated conditions. The present invention relates to isolated DNA which encodes a protein allergen of Ds£jnat££hag£i ^ de£ (I ) . ) house dust mite) or a peptide which includes at least one epitope of a protein allergen of a house dust mite of the genus D rm ^££h g£j.d £. It particularly relates to DNA encoding major allergens of the species D_. arinae , designated Der_f I and Der_f II, or portions of these major allergens (i.e. , peptides which include at least one epitope of Der_f I or of Der_f II) . It also particularly relates to DNA encoding major allergens of D^ E£ ££BZ iHH • designated D«ϊr_£ I and Der_£ II, or portions of these major allergens (i.e. , peptides which include at least one epitope of D£r_£ I or of Der_£ II . The present invention further relates to proteins and peptides encoded by the isolated 2££2i££E]l ££ide , s. (e.g. , D.. farinae , D^g ero^ £Σ £i∑!H≤) DNA. Peptides of the present invention include at least one epitope of a D^_far.inae aller¬ gen (e.g. , at least one epitope of D r_f I or of Der f - - ) or aC least one epitope of a D_. £ erony sinus allergen (e.g. , at least one epitope of D r_ I or of D r_£ II) . It also relates to antibodies speci¬ fic for D_. arin proteins or peptides and to antibodies specific for D L _R££££2Σ£ i2H£ proteins or peptides. D££E .££El2££i£if*£ DNA, proteins and peptides of the present invention are useful for diagnostic and therapeutic purposes. For example, isolated D^ fari.nae protein or peptide can be used to detect sensitivity in an individual to house dust mites and can be used to treat sensitivity (reduce sensitivity or desensitize) in an individual, to whom therapeu- tically effective quantities of the Ω___f rinae protein or peptide is administered. For example, isolated D_ ; farinae protein allergen, such as D _f I or Der_f II can be administered periodically, using standard techniques, to an individual in order to desensitize the individual. Alternatively, a peptide which includes at least one epitope of ^r_f I or of D r_f II can be administered for this purpose. Isolated D_. EliI£Hϊ £Ϊ2H£ protein allergen, such as Der_ I or £_ II, can be administered as described for Der f I or Der f II. Similarly a peptide which includes at least one D≥r £ I epitope or at least one Der_£ II epitope can be administered for this purpose. A combination of these proteins or peptides (e.g. , Der_f I and I r_f ~ - i H££_E - an<i ^ £_E ~^ 'ι or a mixture of both I>£r_j: and D r_£ proteins) can also be administered. The ' use of such isolated proteins or peptides provides a means of desensitizing individuals to important house dust mite allergens. Brief Description of the Drawings Figure 1 is a restriction map of the cDNA insert of clone λgtll f 1, including a schematic representation of the strategy of DNA sequencing. Arrows indicate directions in which sequences were read. Figure 2 is the nucleotide sequence and the predicted amino acid sequence of cDNA λgtll f 1. Numbers above are nucleotide positions; numbers to the left are amino acid positions. Positive amino acid residue numbers correspond to the sequence of the mature excreted Der_f I beginning with threonine. Negative sequence numbers refer to the signal peptide and the proenzyme regions of Der_f I. The arrows indicate the beginning of the proenzyme sequence and the mature D r_f I, respectively. The underlined residues -81 to -78 make up the proposed cleavage site for the proenzyme formation, while the underlined residues 53-55 represent a potential N-glycosylation site. The termination TGA codon and the adjacent polya- denylation signal are also underlined. Amino acid residues 1-28 correspond to a known tryptic peptide sequence determined by conventional amino acid sequencing analysis. Figure 3 is a composite alignment of the amino acid sequences of the mature D<≥r_£ I and D££_f I proteins. The numbering above the sequence refers to Der_£ I. The asterisk denotes the gap that was introduced for maximal alignment. The symbol (.) is used to indicate that the amino acid residue of D r f I at that position is identical to the correspon¬ ding amino acid residue of D £_£ I. The arrows indicate those residues making up the active site of Der_£ I and Per f I . Figure 4 is a comparison of the amino acid sequence in the pre- and pro-peptide regions of Der f I with those of rat cathepsin H, rat cathepsin L, papain, aleurain, CP1, CP2 , rat cathepsin B, CTLA-2, MCP, Der_£ I and actinidin. Gaps, denoted by dashes, were added for maximal alignment. Double asterisks denote conserved amino acid residues which are shared by greater than 80% of the proenzymes; single asterisks show residues which are conserved in greater than 55% of the sequences. The symbol (.) is used to denote semiconserved equivalent amino acids which are shared by greater than 90% of the proenzyme regions. Figure 5 is a hydrophilicity plot of the D£r_£ I mature protein and a hydrophilicity plot of the Der_f I mature protein produced using the Hopp-Woods algorithm computed with the Mac Vector Sequence Analysis Software (IBI, New Haven) using a 6 residue window. Positive values indicate relative hydrophilicity and negative values indicating relative hydrophobicity. Figure 6 is the nucleotide sequence and the predicted amino acid sequence of Der_f II cDNA. Numbers to the right are nucleotide positions and numbers above are amino acid residues. The stop (TAA) signal is underlined. The first 8 nucleotides are from the oligonucleotide primer used to generate the cDNA, based on the Der_£ II sequence. Figure 7 is the nucleotide sequence and predicted amino acid sequence of cDNA λgtll pl(13T). Numbers to the right are nucleotides and numbers above are amino acid positions. Positive amino acids correspond to the sequence of mature excreted U E_E - beginning with threonine. Figure 8 is a restriction map of Der_ f II cDNA, which was generated by computer from the sequence data. A map of De_r_£ II similarly generated is shown for comparison. There are few common restric- tion enzyme sites conserved. Sites marked with an asterisk were introduced by cloning procedures. Figure 9 shows the alignment of Der_f II and Der_£ II cDNA sequences. Numbers to the right are nucleotide position and numbers above and amino acid residues. The top line gives the De:_£ II nucleo¬ tide sequence and the second the Der_£ II amino acid residues. The next two lines show differences of Dejτ_f II to these sequences. Figure 10 is a hydrophilicity plot of Der_f II and De£_£ II using the Hopp-Woods algorithm computed with the Mac Vector Sequence Analysis Software (IBI, New Haven) using a 6-residue window. Figure 11 is a composite alignment of the amino acid sequences of five Der p I clones (a)-(e) which illustrates polymorphism in the Der p I protein. The numbering refers to the sequence of the Der p 1(a) clone. The symbol (-) is used to indicate that the amino acid residue of a Der p I clone is identical to the corresponding amino acid residue of Der p 1(a) at that position. The amino acid sequences of these clones indicates that there may be significant variation in Der p I, with five polymorphic amino acid residues found in the five sequences. £Ξ U£^_2££££iE∑ϊ££_££_£ll£_ΣSZ££^ϊ£ϊl The present invention relates to a nucleotide sequence coding for an allergen from the house dust mite De:m t:o£ha££ide and to the encoded £ £I≥ i£EΪl ££i^£ protein or peptide which includes ■ at least one epitope of the Dermatophagoides allergen. It particularly relates to a nucleotide sequence capable of expression in an appropriate host of a major allergen of I farinae , such as D r f I or Der_f II, or of a peptide which includes at least one epitope of Der_f I or of D r_f II. It also particularly relates to a nucleotide sequence capable of expression in an appropriate host of a major allergen of D_. pteronyssinus , such as Per__£ I or £≤. -E * > or ° - - peptide which includes at least one epitope of I>£r_£ I or of ]>££_£ II. The 2£Σ51£££EΪl S£ϊ^£ nucleotide sequence is useful as a probe for identifying additional nucleotide sequences which hybridize to it and encode other mite allergens, particularly D Λ _farinae or _^ E£ r£n £ i , ιιu allergens. Further, the present invention relates to nucleotide sequences which hybridize to a D^farinae protein-encoding nucleotide sequence or a ^gteron^sinus protein-encoding nucleotide sequence but which encode a protein from another species or type of house dust mite, such as P^ , _]Hi££££ £a (e.g. , D r_m I and Der_m II) . The encoded Dermatoghago^des mite allergen or peptide which includes at least one e£i£a £hag£ide . £ (Der_f I or Der_f II; Der_£ I or Der_2 II) epitope can be used for diagnostic purposes (e.g. , as an 04445 . o_ antigen) and for therapeutic purposes (e.g. , to desensitize an individual). Al ernatively, the encoded house dust mite allergen can be a protein or peptide, such as a IK si££££ ra£ protein or peptide, which displays the antigenicity of or is cross-reac¬ tive with a De:_f or a Der_£ allergen; generally, these have a high degree of amino acid homology. Accordingly, the present invention also relates to compositions which include a Dermatophagoides allergen (e.g. , P^r_f I or allergen, P.er_f II allergen; Deιr_£ I or Per_£ II allergen or other IK_ allergen cross-reactive therewith) or a peptide which includes at least one epitope of a 5ermat££hajg£i.d allergen (D £_f I, Der_f II, D r_£ - > S. —2 - I or - --- -- H__ allergen cross-reactive therewith) individually or in combination, and which can be used for therapeutic applications (e.g. , desensitization) . As is described below, DNA coding for major allergens from house dust mites have been isolated and sequenced. In particular, and • s is described in greater detail in the Examples, two cDNA clones, coding, respectively, for Der_f I allergen and D , er_f II allergen, have been isolated and sequenced. The nucleotide sequences of Der_£ I and Der_£ II have also been determined. The nucleo¬ tide sequence of each of these clones has been compared with that of the homologous allergen from the related mite j ._E£eronχs£.nus (Per_£ I and Der_£ II, respectively), as has the predicted amino acid sequence of each. The following is a description of isolation and sequencing of the two cDNA clones coding for Der f allergens and their comparison with the corresponding ϊ__ E£ £££X iHii allergen and a description of use of the nucleotide sequences and encoded products in a diagnostic or a therapeutic context. £i ^i££.££^_ 3H ££ _ £ iy£Ϊ _ ^_^ £— 11—2- A cDNA clone coding for ^£r_f I, a major allergen from the house dust mite IK f £ill£ > has been isolated and sequenced. A restriction map of the cDNA insert of the clone is represented in Figure 1, as is the strategy of DNA sequencing. This Der_f I cDNA clone contains a 1.1-kb cDNA insert encoding a typical signal peptide, a pro¬ enzyme region and the mature De_r_f I protein. The product is 321 amino acid residues: a putative 18 residue signal peptide, an 80 residue proenzyme (pro-peptide) region, and a 223 residue mature enzyme region. The derived molecular weight is 25,191. The nucleotide sequence and the predicted amino acid sequence of the De:_f I cDNA are repre¬ sented in Figure 2. The deduced amino acid sequence shows significant homology to other cysteine pro¬ teases in the pro-region, as well as in the mature protein. Sequence alignment of the mature De_r_f I protein with the homologous allergen Der_£ I from the related mite D_^_E££r££γ £.inu (Figure 3) revealed a high degree of homology (81%) between the two proteins, as predicted by previous sequencing at the protein level. In particular, the residues com- prising the active site of these enzymes were conserved and a potential -glycosylation site was present at equivalent positions in both mite allergens . Conserved cysteine residue pairs (31, 71) and (65, 103) , where the numbering refers to Der_£ I, are apparently involved in disulphide bond formation on the basis of the assumed similarity of the three dimensional structure of Per_£ I and P£r_f I to that of papain and actinidin, which also have an addi¬ tional disulphide bridge. The fifth and final cysteine residue for which there is a homologous cysteine residue in papain and actinidin is the active site cysteine (residue 35 in £r_f I) . It is not unlikely that the two extra cysteine residues present in ]) £_£ I and Per_f I may be involved in forming a third disulphide bridge. The potential N-glycosylation site in D£r_£ I is also present at the equivalent position in D £_f I, with conservation of the crucial first and last residues of the tripeptide site. The degree of glycosylation of Dβr_f I and Der_£ I has yet to be determined. Carbohydrates, including mannose, galactose, N-acetylglucosamine and N-acetylgalacto- sa ine, have been reported in purified preparations of these mite allergens (Chapman, M.D. , J.^.Iraπ nol^ t 125:587-592 (1980); Wolden, S. et_a1. , Int ._Arch^ Ali£r _ • _ ■ _A££1 i _Immuno I ^ , 6£S : 144-151 (1982)) . Given the degree of homology over the first thirty N-terminal amino acid residues between mature D r_£ I and D£r_m I (70%) and mature De:_f I and Der - (97%) with the D r_m I residues determined by' conventional amino acid sequencing (Platts -Mills TAE ££_ !• > n: W_il£_Alj 1 £ 5Xj._a_Worl.d_ 1 Wi.de_Probl. m, 27-29 (1988); Lind , P. and N.-Horn, In: Mite A11£rg^ x _a_ or1d^ .de_Pr££1em, 30-34 (1988)), it is probable that the full mature D r_m I sequence will confirm an overall 70-80% homology between the Group I mite allergens . Der_m I is an allergen from D_^ mlcr£ r £. High homology between the proenzyme moieties of Der_£ I and Der_f I (91%) over the residues -23 to -1 and the structural analysis of D r_f I suggests that the Group I allergens are likely to have N-terminal extension peptides of the mature protein of homologous structure and, at least for the pro-peptide, composition. Studies on the fine structure of the design of signal sequences have identified three structurally dissimilar regions so far: a positively charged N- terminal (n) region, a central hydrophobic (h) region and a more polar C-terminal (c) region that seems to define the cleavage site (Von Heijne, G. , EMBO_J x , 3:2315-2323 (1984); Eur^_K_Bi£che , 111 -' 1 7 - 2 1 (1983); ^_ £l^_Biol^., 184:99-105 (1985)). Analysis of the signal peptide of Der_f I revealed that it, too, contained these regions (Figure 4). The n-region is extremely variable in length and composition, but its net charge does not vary appreciably with the overall length, and has a mean value of about +1.7. The n-region of the Der_f I signal peptide, with a length of two residues, has a net charge of +2 contributed by the initiator methionine (which is unformylated and hence posi- tively charged in eukaryotes) and the adjacent lysine (Lys) residue. The h-region of Der_f I is enriched with hydrophobic residues, the characteristic feature of this region, with only one hydrophilic residue serine (Ser) present which can be tolerated. The overall amino acid composition of the Der__f I c-region is more polar than that of the h-region as is found in signal sequences with the h/c boundary located between residues -6 and -5, which is its mean position in eukaryotes. Thus, the D r_f I pre-peptide sequence appears to fulfill the requirements to which a functional signal sequence must conform. While the signal sequence of D£r_f I and other cysteine proteases share structural homology, all being composed of the n,h and c-regions, they are highly variable with respect to overall length and amino acid sequence, as is clear in Figure 4. However, significant sequence homology has been shown between the pro-regions of cysteine protease precursors (Ishidoh, K. JC_a].. , FEB^_L. ^£ r , 2 ^ 2^:33-37 (1987)). Alignment of the proenzyme regions of Der_f I and a number of other cysteine proteases (Figure 4) indicated that these proregions share a number of very conserved residues as well as semi-conserved residues which were present in over half of the sequences. This homology was increased if conservative amino acids such as valine (Val) , isoleucine (lie) and leucine (Leu) (small hydropho¬ bic residues) or arginine (Arg) and Lys (positively charged residues) were regarded as identical. The Der_f I proregion possessed six out of the seven highly conserved amino acids and all the residues at sites of conservative changes. The homology at less conserved sites was lower. Homology in the pro- peptide, in particular the highly conserved resi¬ dues, may be important when considering the function of the pro-peptide in the processing of these 5 enzymes, since it indicates that these sequences probably have structural and functional similari¬ ties. Highly cross-reactive B cell epitopes on Der_f I and Der_£ I have been demonstrated using anti- 0 bodies present in mouse, rabbit and human sera (Heymann, P.W. et_a1. , J^Imm nol^ 132:2841-2847 (1986); Platts-Mills , TAE et_al. , J^Allerg^CljLn^ l!2H2£k. 2ϋ :398 " 407 (1986)). However, species- specific epitopes have also been defined in these 15.systems. Murine monoclonal antibodies bound pre¬ dominantly to species-specific determinants (Platts-Mills TAE et_al. , J Λ _Imπuno1^, 1.3£: 1479 -1484 (1987) . Some 40% of rabbit anti-Der_£ I reactivity was accounted for by epitopes unique to D i:_£ I 20 (Platts-Mills, TAE et_al., J^Allergy ^ CljLn^ IS2HS£ - 2^:398-407 (1986)), and some species- specific binding of antibodies from allergic humans was observed, although the majority bind to cross-reactive epitopes (Platts-Mills TAE e al. , J^ 25 ISΪ£HS£I .. I2£: 1479-1484 (1987). The recombinant DNA strategy of gene fragmenta¬ tion and expression was used (Greene, W.K. et_a1. , Ii5!5H££li (1990)) to define five antigenic regions of recombinant Der_£ I which contained B cell epitopes 30 recognized by a rabbit anti-Der_£ I antiseru . Using the technique of immunoabsorption , three of these putative epitopes were shown to be shared with P_ r_f I (located on regions containing amino acid residues 34-47, 60-72 and 166-194) while two appeared to be specific for D£r_£ I (regions 82-99 and 112-140). Pifferences in the reactivity of these peptides to rabbit anti- _ 1 _f ri.na supported the above division into cross-reactive and species- specific epitopes. The sequence differences shown between the Der_£ I and the Der_f I proteins are primarily located in the N and C terminal regions , as well as in an extended surface loop (residues 85-136) linking the two domains of the enzyme that includes helix D (residues 127-136), as predicted from the secondary and tertiary structures of papain and actinidin (Baker, E.N. and J. Drenth, In : ^i£i££i i_ϋ£ ∑£S£i £ϋi £_ E^_^ £I5 iϊ • Vol. 3 , pp. 314-368, John Wiley and Sons, NY. (1987)). The surface location of these residues is supported by the hydrophilicity plots of Iλer_£ I and .D.er_f I in Figure 5, which illustrate the predominantly hydrophilic nature of this region that predicts surface exposure. This region also contains the two species-specific B cell epitopes recognized by the rabbit anti-Der_£ I serum (see above) . Analysis of the sequences in the regions containing the cross- reactive epitopes showed that two of the cross-reac¬ tive epitopes (located in regions 34-47 and 60-72) are completely conserved between Der_£ I and D r_f I, while the majority of residues in a third cross- reactive epitope -containing region (residues region 166-194) were conserved. Expression of results in production of pre- pro-D r_f I protein in E. ££JLi. a recombinant protein of greater solubility, stability and antigenicity than that of recombinant D r_£ I. Protein encoded by Der_f I cDNA has been expressed using a pGEX vector and has been shown by radioimmune assay to react with rabbit anti-IK fari.na antibodies. The availability of high yields of soluble H _~___ I allergen and antigenic derivatives will facilitate the development of diagnostic and therapeutic agents and the mapping of B and T cell antigenic determinants . With the availability of the complete amino acid sequence of recombinant Der_f I, mapping of the epitopes recognized by both the B and T cell com¬ partments of the immune system can be carried out. The use of techniques such as the screening of overlapping synthetic peptides, the use of mono¬ clonal antibodies and gene fragmentation and expression should enable the identification of both the continuous and topographical epitopes of D £_f I. It will be particularly useful to determine whether allergenic (IgE-binding) determinants have common features and are intrinsically different from antigenic (IgG-binding) determinants and whether T cells recognize unique epitopes different from those recognized by B cells. Studies to identify the Der f I epitopes reactive with mite allergic human IgE antibodies and the division of these into determinants cross-reactive with De£_£ I and determinants unique to I) r_f I can also be carried out. B cell (and T cell) epitopes specific for either species can be used to provide useful diagnostic reagents for determining reactivity to the different mite species, while cross -reacting epitopes are candidates for a common imrauno- therapeutic agent. As described in co-pending Application U.S. Serial No. 458,643, incorporated herein by reference, the molecular cloning of mite allergens resulted in the isolation of a cDNA clone coding for £2 _£ I which contained a 0.8-kb cDNA insert. Sequence analysis revealed that the 222 amino acid residue mature recombinant Der_£ I protein showed significant homology with a group of cysteine proteases, including actinidin, papain, cathepsin H and cathepsin B. 2 £iajci.£n_ nd_S ^ U n£e_Ana^2£i£_£f_D£r_f_I ^ 2 A cDNA clone coding for Der_f II, a major allergen from the house dust mite IK farjLnae , has been isolated and sequenced, as described in Example 2. The nucleotide sequence and the predicted amino acid sequence of the Der_f II cDNA are represented in Figure 6. A restriction map of the cDNA insert of a clone coding for De£_f II is represented in Figure 8. Figure 9 shows the alignment of Der_f II and D r_£ II cDNA sequences. The homology of the sequence of Der f II with Der_£ II (88%) is higher than the 81% homology found with Der_£ I and D £_f I, which is significantly 2 different (p<0.05) using the ehi distribution. reason for this may simply be that the Group I allergens are larger and each residue may be less critical for the structure and function of the molecule. It is known, for example, that assuming they adopt a similar conformation to other cysteine proteases, many of the amino acid differences in ^e. . £ I and D r_f I lie in residues linking the two domain structures of the molecules. The 6 cysteine molecules are conserved between the group II allergens, suggesting a similar disulphide bonding, although this may be expected, given the high overall homology. Another indication of the conservation of these proteins is that 34/55 of the nucleotide changes of the coding sequence are in the third base of a codon, which usually does not change the amino acid. Residues that may be of importance in the function of the molecule are Ser 57 where all three bases are changed but the amino acid is conserved. A similar phenomenon exists at residue 88, where a complete codon change has conserved a small aliphatic residue. Again, like D r_£ II, the Der_f II cDNA clone does not have a poly A tail, although the 3' non-coding region is rich in adenosine and has two possible polyadenylation signals ATAA. The nucleotides encoding the first four residues are from the PCR primer which was designed from the known homology of Der_£ II and D r f II from N-terminal amino acid sequencing. A primer based on the C-terminal sequence can now be used to determine these bases, as well as the signal sequence . U e _£f_£he_ ubj_ ££_al L l. rg nj.£_ r££ in£/£e£tide _and £H _ B££^i£S_£ I£ The materials resulting from the work described herein, as well as compositions containing these materials, can be used in methods of diagnosing, treating and preventing allergic responses to mite allergens, particularly to mites of the genus ^ £S ££El £ i^£ • su ch as ^_fari.nae and o her species (e.g. , IK E£££££Σ££i∑lH£ - n - I ._Ei£Ξ££££ £) • In addition, the cDNA (or the mRNA from which it was transcribed) can be used to identify other similar sequences. This can be carried out, for example, under conditions of low stringency and those sequences having sufficient homology (generally greater than 40%) can be selected for further assessment using the method described herein. Alternatively, high stringency conditions can be used. In this manner, DNA of the present invention can be used to identify sequences coding for mite allergens having amino acid sequences similar to that of Der_f I, Der ^ f II, Per_£ I or Per_£ II. Thus, the present invention includes not only P £_f and Per_£ II, but other mite allergens as well (e.g. , other mite allergens encoded by DNA which hybridizes to DNA of the present invention) . Proteins or peptides encoded by the cDNA of the present invention can be used, for example, as "purified" allergens. Such purified allergens are useful in the standardization of allergen extracts or preparations which can be used as reagents for the diagnosis and treatment of allergy to house dust mites. Through use of the peptides of the present invention, allergen preparations of consistent, well-defined composition and biological activity can be made and administered for therapeutic purposes (e.g. , to modify the allergic response of a house dust mite-sensitive individual). Der_f I or De:_f II peptides or proteins (or modified versions thereof, such as are described below) may, for example, modify B-cell response to D _f I or Der_f II, T-cell response to P£r_f I and Per_f II, or both responses. Similarly, £ £_£ I or Der_£ II proteins or peptides may be used to modify B-cell and/or T-cell response to Der_£ I or r_£ II. Purified allergens can also be used to study the mechanism of immunotherapy of allergy to house dust mites, particularly to Der__f I, Der f II, Der_£ I and Der_£ II, and to design modified derivatives or analogues which are more useful in immunotherapy than are the unmodified ("naturally-occurring") peptides. In those instances in which there are epitopes which are cross-reactive , such as the three epitopes described herein which are shared by Der_f I and D r £ I, the area(s) of the molecule which contain the cross-reactive epitopes can be used as common immunotherapeutic peptides to be administered in treating allergy to the two (or more) mite species which share the epitope. For example, the cross-reactive epitopes could be used to induce IgG blocking antibody against both allergens (e.g. , Der f I and Der_£ I allergen) . A peptide containing a univalent antibody epitope can be used, rather than the entire molecule, and may prove advantagious because the univalent antibody epitope cannot crosslink mast cells and cause adverse reactions during desensitizing treatments. It is also possible to attach a B cell epitope to a carrier molecule to direct T cell control of allergic responses . Alternatively, it may be desirable or necessary to have peptides which are specific to a selected D £5a.t£ h g .ide. allergen. As described herein, two epitopes which are apparently D r_ I-specific have been identified. A similar approach can be used to identify other species-specific epitopes (e.g. , Der £ I or II, D_ r_f I or II. The presence in an individual of antibodies to the species-specific epitopes can be used as a quick serological test to determine which mite species is causing the allergic response. This would make it possible to specifically target therapy provided to an individual to the causative species and, thus, enhance the therapeutic effect. Work by others has shown that high doses of allergens generally produce the best results (i.e. , best symptom relief). However, many people are unable to tolerate large doses of allergens because of allergic reactions to the allergens. Modifica- tion of naturally-occurring allergens can be designed in such a manner that modified peptides or modified allergens which have the same or enhanced therapeutic properties as the corresponding naturally-occurring allergen but have reduced side effects (especially, anaphylactic reactions) can be produced. These can be, for example, a peptide of the present invention (e.g. , one having all or a portion of the amino acid sequence of D«jr_f I or Der f II, D r_£ I or Der_£ II). Alternatively, a combination of peptides can be administered. A modified peptide or peptide analogue (e.g., a peptide in which the amino acid sequence has been altered to modify immunogenicity and/or reduce allergenicity or to which a component has been added for the same purpose) can be used for desensitization therapy. Administration of the peptides of the present invention to an individual to be desensitized can be carried out using known techniques . A peptide or combination of different peptides can be administered to an individual in a composition which includes, for example, an appropriate buffer, a carrier and/or an adjuvant. Such compositions will generally be administered by injection, inhalation, transdermal application or rectal administration. Using the information now available, it is possible -° design a Der_f I or Der f II peptide which, when administered to a sensitive individual in sufficient quantities, will modify the individual's allergic response to a Per_ f I and/or Per_f II. This can be done, for example, by examining the structures of Per_ ^ f I or Per_f II, producing peptides to be examined for their ability to influence B-cell and/or T-cell responses in house dust mite-sensitive individuals and selecting appropriate epitopes recognized by the cells. Synthetic amino acid sequences which mimic those of the epitopes and which are capable of down regulating allergic response to Der_f I or Der_f II allergen can be made. Proteins, peptides or antibodies of the present invention can also be used, in known methods, for detecting and diagnosing allergic response to Der_f I or De j r_f II. For example, this can be done by combining blood obtained from an individual to be assessed for sensitivity to one of these allergens with an isolated allergenic peptide of house dust mite, under conditions appropriate for binding of or stimulating components (e.g. , antibodies, T cells, B cells) in the blood with the peptide and determining the extent to which such binding occurs. The D£r_£ I and D_ r_£ II proteins or peptides can be used in a similar manner for desensitization and diagnosis of sensitivity. ___.____. and D\er_£ proteins or peptides can be administered together to treat an individual sensitive to both allergen types. It is now also possible to design an agent or a drug capable of blocking or inhibiting the ability °^ m-H—i. or ^ £_f 11 to induce an allergic reac¬ tion in house dust mite-sensitive individuals. Such agents could be designed, for example, in such a manner that they would bind to relevant anti-Der_f I or anti-D r_f II IgEs, thus preventing IgE-allergen binding and subsequent mast cell degranulatio . Alternatively, such agents could bind to cellular components of the immune system, resulting in suppression or desensitization of the allergic response to these allergens. A non-restrietive example of this is the use of appropriate B- and T-cell epitope peptides, or modifications thereof, based on the cDNA/protein structures of the present invention to suppress the allergic response to D£r_f I or D r_f II allergens. This can be carried out by defining the structures of B- and T-cell epitope peptides ' which affect B- and T-cell function in in vitro studies with blood cells from D£r_f I or Der_f II-sensitive individuals. This can also be applied to Der_£ I or Der_£ II, in order to block allergic response to these allergens. The cDNA encoding Per_f I or P£r_f II or peptide including at least one epitope can be used to produce additional peptides, using known techniques such as gene cloning. A method of producing a protein or a peptide of the. present invention can include, for example, culturing a host cell containing an expression vector which, in turn, contains PNA encoding all or a portion of a selected allergenic protein ' or peptide (e.g., Per _f I, Der_f II or a peptide including at least one epitope) . Cells are cultured under conditions appropriate for expression of the DNA insert (production of the encoded protein or peptide) . The expressed product is then recovered, using known techniques. Alternatively, the allergen or portion thereof can be synthesized using known mechanical or chemical techniques. As used herein, the term protein or peptide refers to proteins or peptides made by any of these techniques. The resulting peptide can, in turn, be used as described previously. DNA to be used in any embodiment of this invention can be cDNA obtained as described herein or, alternatively, can be any oligodeoxynucleotide sequence having all or a portion of the sequence represented in Figures 2 and 6, or their functional equivalent. Such oligodeoxynucleotide sequences can be produced chemically or mechanically, using known techniques. A functional equivalent of an oligonucleotide sequence is one which is capable of hybridizing to a complementary oligonucleotide sequence to which the sequence (or corresponding sequence portions) of Figures 2 and 6 hybridizes and/or which encodes a product (e.g. , a polypeptide or peptide) having the same functional characteristics of the product encoded by the sequence (or corresponding sequence portion) represented in these figures. Whether a functional equivalent must meet one or both criteria will depend on its use (e.g. , if it is to be used only as an oligoprobe, it need meet only the first criterion and if it is to be used to produce house dust mite allergen, it need only meet the second criterion) . The structural information now available (e.g. , DNA, protein/peptide sequences) can also be used to identify or define T cell epitope peptides and/or B cell epitope peptides which are of importance in allergic reactions to D^farinae allergens and to elucidate the mediators or mechanisms (e.g. , interleukin-2 , interleukin-4 , gamma interferon) by which these reactions occur. This knowledge should make it possible to design peptide-based house dust mite therapeutic agents or drugs which can be used to modulate these responses. The present invention will now be further illustrated by the following Examples, which are not intended to be limiting in any way. EXAMPLE^ l££l££i££_£B^_2^SIS££££il££i £-£l-~----£-i£5-- - Der_f_I MATERIALS „ AND METHODS ^££Ξ ^£E^££i^ £_^ £!.££ culture Mites were purchased from Commonwealth Serum Laboratories, Parkville , Australia. Construction of the D.farinae cDNA λgtll library Polyadenylated mRNA was isolated from live IK_ farinae mites and cDNA was synthesized by the RNase H method (Gubler, V. and B.J. Hoffman, Gene, 21:263-269 (1983)) using a kit (Amersham Inter¬ national, Bucks.). After the addition of EcoRI linkers (New England Biolabs, Beverly, MA) the cDNA was ligated to alkaline phosphatase treated λgtll arms (Promega, Madison, WI). The ligated DNA was packaged and plated in E. coli Y1090 (r-) to produce 4 a library of 2x10 recombinants . cDNA_λgtll_]Li.brarγ Screening of the library was performed by hybridization with two probes comprising the two Der £ I cDNA BaraHI fragments 1-348 and 349-857 generated by BamHI digestion of a derivative of the Dej:_£ I cDNA which has had two BamHI restriction sites inserted between amino acid residues -1 and 1 and between residues 116 and 117 by site-directed mutagenesis (Chua, K.Y. .t_al. , J_. . 162:175-182 (1988)). The probes were radiolabelled " ~ 32 with P by nick translation. Phage were plated at 20,000 pfu per 150mm petri dish and plaques were lifted onto nitrocellulose (Schleicher and Schull, Dassel, FRG), denatured and baked (Maniatis , T ._e_t ϋ • • £i ϋi Σ_£ .£BiS£ _i ^£Σ ^£ΣY_ £H i> C ol d Spring Harbor Laboratory Press (1982)). Prehybridizations were performed for 2 hours at 42*C in 50% formamide/5 x SSCE/1 x Denhardt' s/poly C (0. lmg/ml)/poly U(0.1mg/ml) with hybridization overnight at 42*C at 10 cpm/ml. Post hybridization washes consisted of 15 min washes at room temperature with 2 x sodium chloride citrate (SSC)/0.1% sodium dodecylsulphate (SDS), 0.5 x SSC/0.1% SDS, 0.1 x SSC/0.1% SDS successively and a final wash at 50 * C for 30 min in 0.1 x SSC/1% SDS. l££l £Ϊ£2_£f_£NA_from_λgt2i_f_I_cPNA_c2£ne£ Phage DNA from λgtll f 1 clones was prepared by a rapid isolation procedure. Clarified phage plate lysate (1 ml) was mixed with 270 μL of 25% wt/vol polyethylene glycol (PEG 6000) in 2.5M NaCl and incubated at room temperature for 15 min. The mixture was then spun for 5 min in a microfuge (Eppendorf, FRG), and the supernatant was removed. The pellet was dissolved in 100 μL of lOmM Tris/HCl pH8.0 containing 1 mM EDTA and 100 mM NaCl (TE). This DNA preparation was extracted with phenol/TE, the phenol phase was washed with 100 μi TE, the pooled aqueous phases were then extracted another 2 times with phenol/TE, 2 times with Leder phenol (phenol/chloroform/isoamylalcohol; 25:24:1), once with chloroform and the DNA was precipitated by ethanol . 5 _££ H££ i£S To obtain clones for DNA sequence analysis, the λgtll fl phage DNA was digested with EcoRI restriction enzyme (Pharmacia, Uppsala, Sweden) and the DNA insert was ligated to EcoRI-digested M13-derived sequencing vectors mplδ and mpl9 (Maniatis, T. et_ 1. , o1ecu1 ^ ar_C1on^n j g _ A i fe££ ££ΞΣ_ϊ SH i» Cold Spring Harbor Laboratory Press (1982)) . Transformation was carried out using E. ££].£ TG-1 and sequencing was performed by the dideoxynucleotide chain termination method (Sanger, F - £_ !• • roc^Katl^Ac d. Sci. USA, ~ :5463-5467 (1977)) using the Sequenase version 2.0 DNA sequencing kit (U.S.B., Cleveland, Ohio). £iZI°£ϊ £ _ h i£_.reacti£n__ PCR PCR was performed by the Taq PNA polymerase method (Saiki, R.K. et_a^., Science, 2£ : 8 7-491 (1988)) using the TaqPaq kit (Biotech International, Bentley, WA) and the conditions recommended by the supplier with lOng of target DNA and lOpmol of λgtll primers (New England BioLabs, Beverly, MA). RESULTS l££i i££_£^ ^ £_^_I_£^S _£i£B£ Two clones expressing the major mite allergen Der_f I were isolated from the cDNA λgtll library by their ability to hybridize with both of the Der_£ I cDNA probes (nucleotides 1-348 and 349-857). This approach was adopted because amino acid sequencing had shown high homology (80%) between these two allergens (Thomas, W.R. £t:_a^. , Advance£_in_the_Bi££ci.en£££, ] : 1 39 -147 (1989)) . Digestion of the λgtll fl clone DNA with EcoRI re¬ striction enzyme to release the cDNA insert produced three Der_f I cDNA EcoRI fragments : one approximately 800 bases long and a doublet approximately 150 bases long. The Der_f I cDNA insert was also amplified from the phage PNA by the polymerase chain reaction (PCR) resulting in a PCR product of approximately 1.1-kb. Each P r_f I cPNA fragment was cloned separately into the M13-derived sequencing vectors mpl8 and mpl9 and sequenced. 2 Δ_ £H B££_ 2£i∑ i£ The nucleotide sequence of the Der_f I cDNA was determined using the sequencing strategy shown in Figure 1. The complete sequence was shown to be 1084 bases long and included a 335-base long 5' proximal end sequence, a coding region for the entire native Der_f I protein of 223 amino acids with a derived molecular weight of 25,191 and an 80-base long 3' noncoding region (Fig. 2) . The assignment of the threonine residue at position 1 as the NH.-terrainal amino acid of D , er_f I was based on data obtained by NH.-terminal amino acid sequencing of the native protein and the predicted amino acid sequence of recombinant D r_£ I (Chua, K.Y. £_ ].. , : ._l£E-_Ji ____ , 162:175-182 (1988)). The predicted amino acid sequence of the Der_f I cDNA in the NH.-terminal region matched completely with that determined at the protein level (Figure 2). The complete mature protein is coded by a single open reading frame terminating at the TGA stop codon at nucleotide position 1007-1009. The first ATG codon at nucleotide position 42-44 is presumed to be the translation initiation site since the subsequent sequence codes for a typical signal peptide sequence. Ami o_A Ϊd_ U ces_Analγ J. The amino acid sequence predicted by nucleotide analysis is shown in Figure 2. As shown in the composite alignment of the amino acid sequence of mature Der_£ I and Der_f I (Figure 3), high homology was observed between the two proteins. Sequence homology analysis revealed that the Der_f I protein showed 81% homology with the Der_£ I protein as predicted by previous conventional amino acid sequencing. In particular, the residues making up the active site of Der_£ I, based on those determined for papain, actinidin, cathepsin H, and cathepsin B, are also conserved in the De£_f I protein. The residues are glutamine (residue 29), glycine, serine and cysteine (residues 33-35), histidine (residue 171) and asparagine, serine and 45 - 31- tryptophan (residues 191-193) where the numbering refers to P r_f I. The predicted mature D r_f I amino acid sequence contains a potential N-glyco- sylation site (Asn-Thr-Ser) at position 53-55 which is also present as Asn-Gln-Ser at the equivalent position in D\er_£ I. Analysis of the predicted amino acid sequence of the entire Der_f I cDNA insert has shown that, as for other cysteine proteases (Figure 4) , the P £_f I protein has pre- and proform intermediates. As previously mentioned, the methionine residue at position -98 is presumed to be the initiation methionine. This assumption is based on the fact that firstly, the 5' proximal end sequence from residues -98 to -81 is composed predominantly of hydrophobic amino acid residues (72%), which is the characteristic feature of signal peptides (Von Heijne, G. , EMB0_J^_, 3:2315-2323 (1984)) . Secondly, the lengths of the presumptive pre- (18 amino acid residues) and pro-peptides (80 residues) are similar to those for other cysteine proteases (Figure 4). Most cysteine proteases examined have about 120 preproenzy e residues (of which an average of 19 residues form the signal peptide) with cathepsin B the smallest with 80 (Ishidoh, K. et_a1. , FEBS mi S. , 226 ^ :32-37 (1987)). Per_f I falls within this range with a total of 98 preproenzyme residues. By following the method for predicting signal- sequence cleavage sites outlined in Von Heijne, it is proposed that cleavage from the pre-Der_f I sequence for proenzyme formation occurs at the signal peptidase cleavage site lying between Ala (-81) and Arg (-80) (Von Heijne, G. , Eur i _J_ L ££h S^. 133:17-21 (1988) and ^_M £ 1^_Bi£1^, 184:99-105 (1985)). Thus, the sequence from residues -98 to -81 codes for the leader peptide while the proenzyme moiety of Der_f I begins at residue Arg (-80) and ends at residue Glu (-1). Ex m ^e_ 2££2 i£n_and_^h £ac^ r^za_ti : £n_£f_ PNA_C d^n£_^£r Per_f_ 2 MATER2AL _ANP_METH0P Ami.n£_acj i d_ e uenc _ana2χ£i£ Pr £ ra£jL£n_£f_λ£t^l. ^.fa£ina _cPNA_^i : £ati ^ £n£ D^farinae was purchased from Commonwealth Serum Laboratories, Parkville, Australia, and used to prepare mRNA (polyadenylated RNA) as described (Stewart, G.A. and W.R. Thomas, Int -1 _Arch._ Allergy AEEl_IS5H2£ ..» 83:384-389 (1987)). The mRNA was suspended at approximately 0.5μg/μl and 5μg used to prepare cPNA by the RNase H method (Gubler, U. and Hoffman, B.J. , Gene, 25:263-269 (1983)) using a kit (Amersham International, Bucks). EcoRI linkers (Amersham, GGAATTCC) were attached according to the method described by Huynh £^_a^. , Constructing and screening cPNA libraries in gtlO and gtll , In: Glover, PNA Cloning vol. A practical approach pp. 47-78 IRL Press, Oxford (1985)). The PNA was then digested with EcoRI and recovered from an agarose gel purification by electrophoresis into a PEAE membrane (Schleicher and Schuell, Dassel, FRG, NA-45) according to protocol 6.24 of Sambrook e t al - > (Sambrook et_a3.. , Molecular Cloning, A Laboratory Manual, 2d Ed. , Cold Spring Harbor Laboratory Press (1989)), except 0.5M arginine base was used for elution. The cPNA was then ligated in λgtlO and λgtll at an arms to insert ratio of 2:1. Some was packaged for plaque libraries and an aliquot retained for isolating sequences by polymerase chain reaction as described below. !££ii£ϊ£I!_£.£ ^ £_^_ϋ_£5ϋ _ ∑_ £i∑51 Σ £_ ii i£ ££Ϊ£S To isolate P£r__f II cDNA, an oligonucleotide primer based on the N-terminal sequence of £r_ II was made because their amino acid residues are identical in these regions (Heymann, P.W. e _______ . , _ __ Allergy Clin. Immunol. , 83:1055-1087 (1989)). The primer GGATCCGATCAACTCGATGC-3' was used. The first GGATCC encodes a BamHI site and the following sequence GAT... encodes the first 4 residues of P r E - For -•- - other primer the λgtll TTGACACCAGACC- AACTGGTAATG-3 ' reverse primer flanking the EcoRI cloning site was used (New England Biolabs, Beverly, MA). The Per_£ II primer was designed to have approximately 50-60% G-C and to end on the first or second, rather than the third, base of a codon (Gould, S.J. et_al. , Proc^Natl^_Aca d___" c i^_, j3£:1934-1938 (1989); Summer, R. and P. Tautz , N£c3.eic_Aci.d_R££_ ; _ , 12:6749 (1989)). The PCR reactions were carried out in a final reaction volume of 25μl containing 67mM Tris-HCL (pH8.8 at 25'C), 16.6mM (NH 4 ) 2 S0 > 40μM dNTPs , 5mM 2 -mercaptoethanol , 6μM EDTA, 0.2mg/ml gelatin, 2mM MgCl„, lOpmoles of each primer and 2 units of Taq polymerase. Approximately O.OOlμg of target PNA was added and the contents of the tube were mixed and overlayed with paraffin oil. The tubes were initially denatured at ' 95*C for 6 minutes, then annealed at 55*C for 1 minute and extended at 72 * C for 2 minutes. Thereafter for 38 cycles, denaturing was carried out for 30 seconds and annealing and extension as before. In the final (40th) cycle, the extension reaction was increased to 10 minutes to ensure that all amplified products were full length. The annealing temperature was deliberately set slightly lower than the Tm of the oligonucleotide primers (determined by the formula Tm-69.3 + 0. 1(G+C%) -650/oligo length) to allow for mismatches in the N-terminal primer. 5μl of the reaction was then checked for amplified bands on a 1% agarose gel. The remainder of the reaction mixture was extracted with chloro¬ form to remove all of the paraffin oil and ethanol precipitated prior to purification of the amplified product on a low melting point agarose gel (Bio-Rad, Richmond, CA) . ——£i££inj_£f_PCR_£r£duc The ends of the purified PCR product were filled in a reaction containing 10 mM Tris HCl, 10 mM MgCl 2 , 50 mM NaCl, 0.025 mM dNTP and lμl of Klenow enzyme in a final volume of lOOμl. The reaction was carried out at 37 * C for 15 minutes and heat inactivated at 70°C for 10 minutes. The mixture was Leder phenol extracted before ethanol precipitation. The resulting blunt ended PNA was ligated into M13mpl8 digested with Sjna I in a reaction containing 0.5M ATP, 1 X ligase buffer and 1 unit of T, ligase at 15"C for 24 hrs and transformed into E_. £ U TGI made competent by the Cad ., method. The transformed cells were plated out as a lawn on L + G plates and grown overnight at 37'C. ∑££E £ £ϊ££_£ 1\_ iBiai .L£££ ££ f_ £ _∑ I£E. £ _.££.£ *U.££H£££i£.S Isolated white plaques were picked using an orange stick into 2.5ml of an overnight culture of TGI cells diluted 1 in 100 in 2 X TY broth, and grown at 37 * C for 6 hours. The cultures were pelleted and the supernatant removed to a fresh tube. To a 1ml aliquot of this supernatant 270μl of 20% polyethylene glycol, 2.5M NaCl was added and the tube was vortexed before allowing it to stand at RT for 15 minutes. This was then spun down again and all traces of the supernatant were removed from the tube. The pellet was then resuspended in lOOμl of 1 X TE buffer. At least 2 phenol:TE extractions were done, followed by 1 Leder phenol extraction and a CHCL- extraction. The DNA was precipitated in ethanol and resuspended in a final volume of 20μl of TE buffer. £ϋΔ_ΔE . ∑£i DNA sequencing was performed with the dideoxy- nucleotide chain termination (Sanger, F. al. , pr££^__Nat^_Acad^_Sci^, 2it 5463 " 5467 (1977)) using DNA produced from M13 derived vectors mpl8 and mpl9 in 1• ££ii TG 1 and T4 DNA polymerase ( Sequenase version 2.0, USB Corp. , Cleveland, Ohio; Restriction endonucleases were from Toyobo , (Osaka, Japan). All general procedures were by standard techniques (Sambrook, J. £c_a^. , A Laboratory Manual, 2d Ed. Cold Spring Harbor Laboratory Press (1989)). The sequence analysis was performed using the Mac Vector Software (IBI, New Haven, CT) . RESULTS IK_farina cDNA ligated in λgtll was used to amplify a sequence using an oligonucleotide primer with homology to nucleotides coding for the 4 N terminal residues of D££_£ II and a reverse primer for the λgtll sequence flanking the coding site. Two major bands of about 500 bp and 300 bp were obtained when the product was gel electrophoresed. These were ligated into M13 mpl8 and a number of clones containing the 500 bp fragment were analyzed by DNA sequencing. Three clones produced sequence data from the N-terminal primer end and one from the other orientation. Where the sequence data from the two directions overlapped, a complete match was found. One of the clones read from the N-terminal primer, contained a one-base deletion which shifted the reading frome . It was deduced to be a copying error, as the translated sequence from the other two clones matched the protein sequence for the first 20 amino acid residues of the allergen. The sequence of the clones showing consensus and producing a correct reading frame is shown in Figure 6, along with the inferred amino acid sequence. It coded for a 129 residue protein with no N-glycosylation site and a calculated molecular weight of 14,021 kD. No homology was found when compared to other proteins on the GenBank data base (61.0 release). It did, however, show 88% amino acid residue homology with Der II shown in the alignment in Figure 9. Seven out of the 16 changes were conservative. The conserved residues also include all the cysteines present at positions 8, 21, 27, 73 and 119. There was also considerable nucleotide homology, although the restriction enzyme map generated from the sequence data for commonly used enzymes is different from Der II (Figure 8). The hydrophobicity plots of the translated sequence of Der f II and Der p π shown in Figure 10 are almost identical. It is expected that there are sequence polymorphisms in the nucleic acid sequence coding for Der p I, Der II, Der f I and Der f II, and it will be appreciated by one skilled in the art that one or more nucleotides in the nucleic acid sequences coding for Der p I, Der p II, Der f I and Der f II may vary among individual mites due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of the invention. Amino acid polymorphisms were discovered during the sequencing of different Der p I clones. These polymorphisms are shown in Figure 11. Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims: CLAIMS 1. Isolated DNA encoding a protein allergen of Dermatophagoides farinae. or isolated DNA encoding a peptide which includes at least one epitope of a protein allergen of Dermatophagoides faripae. or isolated DNA which hybridizes with isolated DNA encoding a protein allergen or a peptide of Dermatophagoides __________ 2. Isolated DNA of Claim 1 encoding Der f I. 3. Isolated DNA encoding a protein allergen of Dermatophagoides pteronyssinus. or isolated DNA encoding a peptide which includes at least one epitope of a protein allergen of Dermatophagoides pteronyssinus. or isolated DNA which hybridizes with isolated DNA encoding a protein allergen or a peptide of Dermatophagoides pteronyssinus. 4. Isolated DNA of Claim 3 encoding Der p I. 5. Isolated DNA encoding all or a portion of a protein allergen of the genus Dermatophagoides. the isolated DNA having all or a portion of a nucleotide sequence selected from the group consisting of : the nucleotide sequence of Der f I. as represented in Figure 2; the nucleotide sequence of Der f II, as represented in Figure 6; and the nucleotide sequence of Der p I, as represented in Figure 7. 6. An isolated protein allergen of Dermatophagoides farinae or an isolated peptide which includes at least one epitope of a protein allergen of Dermatophagoides farinae. 7. An isolated protein allergen or an isolated peptide of Claim 6 wherein the protein allergen of Dermatophagoides farinae is Der f I. 8. An isolated protein allergen or an isolated peptide of Claim 6 wherein the protein allergen of Dermatophagoides farinae is Der f II. 9. An isolated protein allergen or an isolated peptide encoded by DNA which hybridizes to isolated DNA encoding a protein allergen or a peptide of Dermatophagoides farinae. 10. A protein allergen or peptide having antigenic cross reactivity and a high degree of homology with a protein allergen or a peptide of Deri I. 11. An isolated protein allergen or an isolated peptide of Claim 7 which is mechanically or chemically synthesized or which is produced in a host cell transformed with isolated DNA of Claim 1. 12. An isolated protein allergen of Der f I or an isolated peptide which includes at least one epitope of a protein allergen of Der f I, the isolated protein or isolated peptide encoded by DNA having all or a portion of the nucleotide sequence of Der f I, as represented in Figure 2. 13. An isolated protein allergen of Per f II or an isolated peptide which includes at least one epitope of a protein allergen of Per f π, the isolated protein or isolated peptide encoded by DNA having all or a portion of the nucleotide sequence of Der f π, as represented in Figure 6. 14. An isolated protein allergen Der p I or an isolated peptide which includes at least one epitope of a protein allergen of Per p I, the isolated protein or isolated peptide encoded by DNA having all or a portion of the nucleotide sequence of Der p I, as represented in Figure 7. 15. A diagnostic reagent comprising an isolated protein allergen of Dermatophagoides farinae or an isolated peptide which includes at least one epitope of a protein allergen of Dermatophagoides farinae. 16. A diagnostic reagent of Claim 15 comprising an isolated peptide which includes at least one epitope of a protein allergen of Der fπ. 17. A diagnostic reagent of Claim 15 wherein the protein allergen of Dermatophagoides farinae is \____j I. 18. A therapeutic composition comprising an isolated protein allergen of Dermatophagoides farinae or an isolated peptide which includes at least one epitope of a protein allergen of Dermatophagoides __________ 19. A therapeutic composition of Claim 18 comprising an isolated peptide which includes at least one epitope of a protein allergen of Der f II. 20. A therapeutic composition of Claim 18 wherein the protein allergen of Dermatophagoides faπnaε is Dgr f I. 21. A therapeutic composition of Claim 18 additionally comprising an isolated protein allergen of Dermatophagoides pteronyssinus having the amino acid sequence of Figure 7, or an isolated peptide which includes at least one epitope of a protein allergen of Dermatophagoides pteronyssinus having a portion of the amino acid sequence of Figure 7 . 22. A therapeutic composition comprising an isolated protein allergen of Dermatophagoides pteronyssinus having the amino acid sequence of Figure 7, or an isolated peptide which includes at least one epitope of a protein allergen of Dermatophagoides pteronyssinus having a portion of the amino acid sequence of Figure 7. 23. A method of treatment for sensitivity in an individual to house dust mites of the species Dermatophagoides farinae. comprising administering to the individual an effective therapeutic amount of an isolated protein allergen of Der f I. Der f II, or a combination thereof. 24. A method of treatment for sensitivity in an individual to house dust mites of the species Dermatophagoides farinae. comprising administering to the individual a sufficient quantity of an isolated peptide of per f I. Per fπ. or a combination thereof, which peptide includes at least one epitope of the protein allergen. 25. A method of detecting sensitivity in an individual to house dust mites of the species Dermatophagoides _____ \ , comprising administering to the individual a sufficient quantity of an isolated protein allergen of Dermatophagoides farinae or an isolated peptide which includes at least one epitope of a protein allergen of Dermatophagoides farinae. and determining the occurrence of an allergic response in the individual to the protein allergen. 26. A method of Claim 25, wherein the protein allergen of Dermatophagoides farinae is Der f I or Der f π. 27. A method of detecting sensitivity in an individual to house dust mites of the species Dermatophagoides farinae. comprising combining a blood sample of the individual with isolated Der f I, Der f π, or both Der f I and Der f π, under conditions appropriate for binding of blood components with Der f I or Der f II, and determining the extent to which such binding occurs. 28. A modified protein allergen of the genus Dermatophagoides or a modified peptide of a protein allergen of the genus Dermatophagoides which, when administered to a house dust mite- sensitive individual, reduces the allergic response of the individual to house dust mite. 29. A modified protein allergen or modified peptide of Claim 28 wherein the protein allergen of the genus Dermatophagoides is Per f l or Der f π. 30. An isolated protein allergen of Dermatophagoides pteronyssinus or an isolated peptide which includes at least one epitope of a protein allergen of Dermatophagoides pteronyssinus, the isolated protein or isolated peptide having all or a portion of the amino acid sequence of βg B I as represented in Figure 11.

Inventors: CHUA KAW-YAN, THOMAS WAYNE ROBERT, CHUA, KAW-YAN, THOMAS, WAYNE, ROBERT
Applicants: WESTERN AUSTRALIAN RES INST, THE WESTERN AUSTRALIAN RESEARCH INSTITUTE FOR CHIL
Application year: 1991
Main classification: C12P21
IPC codes: A61K38, A61K39, A61K49, A61P37, C07K14, C12N15, C12P21, G01N33