Recombinant hybrid allergen constructs with reduced allergenicity that retain immunogenicity of the natural allergen

FIELD OF THE INVENTION

[0002] The present invention is directed to recombinant hybrid proteins having native conformation and containing at least one antigenic peptide sequence introduced into a scaffold protein. The invention is further directed to recombinant nucleic acids and vectors encoding the recombinant vespid hybrid proteins and cells containing the recombinant vectors. Such recombinant hybrid proteins are useful for eliciting an immune response without eliciting an allergenic response, and are therefore particularly useful for therapeutic treatment of allergy.

BACKGROUND OF THE INVENTION

[0003] Genetically predisposed individuals become sensitized (allergic) to antigens originating from a variety of environmental sources, to the allergens of which the individuals are exposed. The allergic reaction occurs when a previously sensitized individual is re-exposed to the same or a homologous allergen. Allergic responses range from hay fever, rhinoconductivitis, rhinitis and asthma to systemic anaphylaxis and death in response to, e.g., bee or hornet sting or insect bite. The reaction is immediate and can be caused by a variety of allergens such as compounds originating from grasses, trees, weeds, insects, food, drugs, chemicals and perfumes.

[0004] Biochemical Aspects of Allergens

[0005] Insect sting allergy to bees and vespids is of common occurrence. The vespids include hornets, yellow jackets and wasps (Golden et al., 1989, Am. Med. Assoc. 262:240). Susceptible people can be sensitized on exposure to minute amounts of venom proteins; as little as 2-10 μg of protein is injected into the skin on a single sting by a vespid (Hoffman and Jacobson, 1984, Ann. Allergy. 52:276).

[0006] There are many species of hornets (genus Dolichovespula), yellowjackets (genus Vespula) and wasp (genus Polistes) in North America (Akre et al., 1980, “Yellowjackets of America North of Mexico,” Agriculture Handbook No. 552, US Department of Agriculture). The vespids have similar venom compositions (King et al., 1978, Biochemistry 17:5165; King et al., 1983, Mol. Immunol. 20:297; King et al., 1984, Arch. Biochem. Biophys. 230:1; King et al., 1985, J. Allergy and Clin. Immunol. 75:621; King, 1987, J. Allergy Clin. Imnmunol. 79:113; Hoffman, 1985, J. Allergy and Cin. Immunol. 75:611). Their venom each contains three major venom allergens, phospholipase (37 kD), hyaluronidase (43 kD) and antigen 5 (23 kD) of as yet unknown biological function.

[0007] In addition to the insect venom allergens described above, the complete amino acid sequence of several major allergens from different grass (Perez et al., 1990, J. Biol. Chem. 265:16210; Ansari et al., 1989, Biochemistry 26:8665; Silvanovich et al., 1991, J. Biol. Chem. 266:1204), tree pollen (Breiteneder, 1989, EMBO J. 8:1935; Valenta et al., 1991, Science, 253:557), weed pollen (Rafnar et al., 1991, J. Biol. Chem. 266:1229; Griffith et al., 1991, Int. Arch. Allergy Appl. Immunol. 96:296), mites (Chua et al., 1988, J. Exp. Med. 167:175), cat dander (Griffith et al., 1992, Gene. 113:263), and mold (Aruda et al., 1990, J. Exp. Med. 172:1529; Han et al., 1991, J. Allergy Clin. Immunol. 87:327) have been reported. These major allergens are proteins of 10-40 kD and they have widely different biological functions. Nearly all allergens of known sequences have a varying extent of sequence similarity with other proteins in our environment. A comprehensive list of nearly all known allergens is maintained under the auspices of the World Health Organization (WHO) and International Union of Immunological Standards (IUIS) Sub-Committee for Allergen Nomenclature, available at Internet site allergen.org on the World Wide Web.

[0008] T and B Cell Epitope of Allergens

[0009] Antibody responses to proteins require the collaboration of T helper and B lymphocytes and antigen presenting cells (APC). The antigen receptors of B cells are the membrane-bound antibody (Ab) molecules, which recognize and bind immunogens directly. The antigen receptors of T cells (TCR) only recognize and bind complexes of antigenic peptide-MHC class II molecule. Immunogens are first processed by APC into peptides that are presented on the surface of APC in association with the MHC class II molecules (Unanue, 1992, Current Opinion in Immunol 4:63). As MHC molecules are highly polymorphic in individuals, they have different specificity of binding antigenic peptides (Rothbard and Gefter, 1991, Ann. Rev. Immunol. 9:527). This is one mechanism for genetic control of immune response.

[0010] T helper cells are activated when the antigen receptor binds the peptide-MHC complex on the surface of APC. Activated T cells secrete lymphokines. In mice (Street and Mosmann, 1991, FASEB J. 5:171) and apparently in humans (Wierenga et al., 1990, J. Immunol. 144:4651; Parronchi etal., 1991, Proc. Natl. Acad. Sci. USA. 88:4538) the T helper cells can be divided into different types on the basis of their patterns of lymphokine production. Primarily, T helper cells divide into two groups: Th1 cells producing IL-2 and IFN-γ and Th2 cells producing IL-4 and IL-5. These lymphokines in turn influence the antigen-activated B cells to differentiate and proliferate into plasma cells secreting Abs of different isotypes. IL-4 is one lymphokine known to influence IgE synthesis (Finkelman et al., 1990, Ann. Rev. Immunol. 8:303).

[0011] It is believed that the entire accessible surface of a protein molecule can be recognized as epitopes by the antigen receptors of B cells, although all epitopes are not necessarily recognized with equal likelihood (Benjamin et al., 1984, Ann. Rev. Immunol. 2:67). B cell epitopes of a protein are of two types: topographic and linear. The topographic type consists of amino acid residues which are spatially adjacent but may or may not be sequentially adjacent. The linear type consists of only sequentially adjacent residues. X-ray crystallographic data of Ag-Ab complexes indicate the size of their complementary binding region to have 16-17 amino acid residues (Amit et al., 1986, Science 233:747). Phospholipase, like other protein antigens, can have both types of B cell epitopes or only one. Vespid antigen 5s have both types. Bee venom melittin appears to have only one B cell epitope of linear type (King et al., 1984, J. Immunol. 133:2668).

[0012] T cell epitopes of proteins consist of only the linear type since they are peptides that have been processed in the lysosomes of APC by proteases (Unanue, 1992, Curr. Op. Immunol. 4:63). Analysis of naturally processed antigenic peptides bound to MHC class II molecules indicates that their size ranges from about 13 to 17 amino acid residues, but analysis of synthetic peptide-MHC class II molecule complex for their T cell proliferate response suggests a minimal size of about 8 amino acid residues (Cf. Rudensky et al., 1991, Nature 353:622). Studies suggest that T cell epitopes are distributed throughout the entire protein molecule, and they may function as major or minor determinants depending on the MHC haplotype of the immunized host (Roy et al., Science 244:572; Gammon et al., 1987, Immunol. Rev. 98:53; O'Hehir et al., 1991, Ann. Rev. Immunol. 9:67).

[0013] Hypersensitivity of the immediate type is known to be caused by the presence of allergen-specific IgE. IgE is found in the circulation and bound to specific IgE-Fc receptors on mast cells and basophils. Cross-linking of cell-bound IgE by allergens leads to release of histamine, leukotrienes and other chemical mediators that cause the allergic symptoms. IgE is one of the different isotypes of immunoglobulins. As pointed out above, lymphokines secreted by T cells influence isotype switch events in B cells.

[0014] Because of the central role of Th2 cells in determining the isotype switch event of B cells, the T cell epitopes of several allergens have been mapped (Cf. O'Hehir et al., supra). These allergens include ragweed Amb III, rye grass Lol p I, cat Fel d I, mouse urine Mus m I, midge Chi t I, bee venom phospholipase A2 (Dhillon et al., 1992, J. Allergy Clin. Immunol. 90:42) and melittin (Fehlner et al., 1991, J. Immunol. 146:799). The data do not reveal any unusual or common structural features. However, any conclusion from these data is qualified as these data are collected from humans and mice of different haplotypes.

[0015] Modulation of T and B Cell Responses

[0016] Normally hosts are tolerant to the dominant B and T cell epitopes of self proteins by clonal deletion and anergy. However this tolerance can be broken under certain circumstances (Gammon et al., 1991, Immunol. Today 12:193; Basten et al., 1991, Immunol. Rev. 122:5). It has been suggested that self-tolerance is broken in autoimmune diseases through encounters with foreign proteins that are similar to host proteins. Therefore the sequence similarity of allergens with autologous proteins is of interest for closer investigation.

[0017] Mature B cells are activated in response to multivalent antigens, which can cross-link cell surface Ig receptors (DeFranco, 1987, Ann. Rev. Cell Biol. 3:143), and they are rendered anergic in response to mono-valent antigen (Basten et al., 1991, supra). Antigen activation of T cells requires not only the integration of TCR with peptide-MHC complex but also with other co-stimulating signals on the surface of APC (Schwartz, 1990, Science 248:1349; Jenkins and Miller, 1992, FASEB J. 6:2428). Interaction of TCR with peptide-MHC complex in absence of co-stimulating signals can lead to T cell anergy.

[0018] Experimental autoimmune encephalomyelitis (EAE) in mice or rats is a well-studied model for multiple sclerosis. Many studies have identified immunodominant T cell determinants for myelin basic protein, which is used to induce this condition. Peptides that correspond to immunodominant epitopes of myelin basic protein can induce tolerance to the same peptide antigen or to the intact myelin basic protein. The same peptides that induced tolerance could also induce T cell anergy in an ongoing autoimmune response (Gaur et al., 1992, Science 259:1491-1494).

[0019] Early studies have shown that the physical state of the immunogen and the route of immunization are important variables in determining the outcome of an immune response. In the light of our current understanding, these variables may well influence antigen presentation so as to have T and B cell activation or anergy.

[0020] Immunotherapy

[0021] One way to treat allergic diseases is by immunotherapy, which involves repeated subcutaneous injections of the offending allergen(s) into patients. For most patients following immunotherapy, allergen-specific IgG levels initially rise. A gradual decrease of allergen-specific IgE levels follows the IgG rise (Norman, 1993, Current Op. Immunol. 5:968). Treated patients also show changes in their T cell cytokine profile: IL-4 and IL-5 levels decreased and IFN-γ level increased (Secrist et al., 1993, J. Exp. Med. 178:2123.)

[0022] Studies have shown that immunotherapy with high doses of allergens is more effective for symptom reduction than that with low doses. However, effective dosages of allergens were limited by the potential danger of unwanted systemic allergic reaction in patients. Because of the undesirable systemic reaction on immunotherapy with native allergens, there has been continued interest in the development of modified allergens with reduced allergenic activities for immunotherapy (T. P. King, 1993, in “Bronchial Asthma,” edited by E. B. Weiss and M. Stein, Little Brown, Boston, pp. 43-49; R. E. O'Hehir et al., 1991, supra).

[0023] Allergenicity depends on the interaction of a multi-valent allergen with basophil or mast cell-bound IgE antibodies. Therefore, allergenicity of a protein can be reduced by decreasing its B cell epitope density. Reduction of B cell epitope density of a protein can be accomplished by several approaches. One approach is by partial or complete denaturation of allergens by chemical treatment or fragmentation (Takatsu et al., 1975, J Immunol 115:1469; Pesce et al., 1990, Int Arch Allergy Appl Immunol 92:88; Vrtala et al., 1997, J Clin Invest 99:1673) since the majority of B cell epitopes are of the discontinuous type, i.e., dependent on the native conformation of proteins. For example, urea treatment of the major allergen from ragweed pollen led to irreversible denaturation with loss of the discontinuous B cell epitopes but retention of the continuous B and T cell epitopes (Takatsu et al., 1975, J Immunol 115:1469). Immunotherapy of patients with the fully denatured ragweed allergen showed no changes in specific IgE and IgG levels for the native allergen although the peripheral blood mononuclear cells of treated patients did show decreased proliferative response on antigen stimulation (Norman et al., 1980, J Allergy Clin Immunol 66:336). Use of partially denatured allergens has also been proposed. This is exemplified by the recombinant mite allergens, which lack the cysteine residues that are involved in maintaining the native structure of the protein (Smith et al., 1996, Mol Immunol 33:399; T. Takai et al., 1997, Nature Biothechnology 15:754).

[0024] Two reports have appeared on the use of T cell epitope peptides to modulate allergen-specific immune responses. One report is on the subcutaneous injection of mice with two peptides from the major cat allergen Fel d I to decrease T cell response to the entire molecule Fel d I (Briner et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:7608-12). Another is on the intranasal therapy with a peptide from the major mite allergen Der p I to suppress allergen-specific response in naive or sensitized mice (Hoyne et al., 1993, J. Exp. Med. 178:1783-1788).

[0025] These findings suggested the use of T cell peptides as immunotherapeutic reagents since T cell peptides are like the denatured allergens in that they lack the discontinuous B cell epitopes. The dominant T cell peptides of several allergens were tested in patients; cytokine level changes but not antibody level changes were observed (Muller et al., 1998, J Allergy Clin Immunol 101:747; Simons et al., 1996, Int Immunol 8:1937; Creticos et al., 1997, J Allergy Clin Immunol 99:401; Marcotte et al., 1997, J Allergy Clin Immunol 99:405). Importantly, these clinical findings with the urea-denatured allergen and T cell peptides suggest that the retention of the discontinuous B cell epitopes as well as the continuous B and T cell epitopes is required for modified allergens to be effective in modulating both antibody and cellular immune responses.

[0026] A second approach to reduce the accessibility of B cell epitopes of allergen involves polymerization of the allergen by formaldehyde or glutaraldehyde treatment (Marsh, 1971, Int Arch Allergy Appl Immunol 41:199; Patterson et al., 1973, J Immunol 110:1413) or by attachment of non-immunogenic polymers (King et al., 1979, J Exp Med 149:424). Glutaraldehyde polymerized antigens were found to be processed differently from the natural antigens in mice, and they were processed by antigen-presenting cells that secrete cytokines promoting Th1 responses (Gieni et al., 1993,. J Immunol 150:302). This second approach for improved immunotherapy had been tried with ragweed pollen allergens with immunological findings similar to those with natural allergens (Norman et al., 1982, J Allergy Clin Immunol 70:248; Norman, 1984, J Allergy Clin Immunol 73:787). One limitation of this approach was that near complete loss of the discontinuous B cell epitopes usually occurred when allergens were modified to achieve greater than 100-fold reduction in allergenicity.

[0027] A third approach is by site-directed mutagenesis to selectively alter the contact amino acid residues of B cell epitopes of allergens. If the key contact residues of B cell epitopes are known, this can be a useful approach. For example, a single residue mutation of Glu to Ser in the major birch allergen abolished its binding of a murine antibody, and resulted in a 40% decrease of its binding of IgEs from a serum pool of allergic patients (Mirza et al., 2000, J Immunol. 165:331). The different decreases probably reflect that the murine antibody and the human IgEs are respectively of monoclonal and polyclonal origins.

[0028] Since an MHC class II molecule of any one haplotype can bind a wide range of peptides in its binding groove, it may be possible to modulate T cell response by inhibition of allergen-derived T cell epitope binding to MHC molecules with other peptides. For example, a mouse lysozyme peptide which is not immunogenic by itself in H-2k mice inhibits T cell response to hen egg white lysozyme (Adorini and Nagy, 1990, Immunol. Today 11:21). Another example is the in vitro inhibition of T cell response to a mite allergen by an influenza HA peptide (O'Hehir et al., 1991, J. Allergy Clin. Immunol. 87:1120).

[0029] Immune response to an immunogen/allergen thus depends in part on the genetic make-up of the host, the route and mode of immunization and the immunogen/allergen. The extent to which an allergen determines the outcome of IgE response is not known. How many B and T cell epitopes must each allergen have? Are immunodominant B or T cell epitopes of an allergen recognized by different or all susceptible individuals? Are there T cell epitopes which favor IgE class switch events in B cells? Does antigenic cross reactivity of allergens with host proteins play a role as to why some proteins are more allergenic than others are? Can tolerance to a multi-valent allergen be induced by treatment with a single or a combination of B or T cell epitopes?

[0030] U.S. Pat. Nos. 5,593,877; 5,612,209, 5,804,201, 6,106,844, 6,270,763 and 6,287,559 and U.S. application Ser. No. 09/166,205 to King disclose the isolation of cDNAs encoding vespid venom proteins and the deduced amino acid sequences of proteins encoded by the cDNAs. The cDNAs allow the expression and purification of large quantities of vespid venom proteins and polypeptides for use in immunotherapy. Sequences, however, fail to yield information on the native structure of vespid venom. Hence, the cDNAs and deduced amino acid sequences do not yield information on discontinuous epitopes. Nor do the deduced vespid venom amino acid sequences predict epitopes that will be present on the surface of recombinantly produced vespid venom proteins. Consequently, the cDNA and deduced amino acid sequences alone cannot accurately predict which regions or peptides of vespid venom proteins will serve as efficient immunogens to stimulate a B cell-mediated immune response. Nor can the cDNA and deduced amino acid sequences alone predict the epitope density on the surface of a vespid venom protein, which is an important determinant of the potential to crosslink surface IgE molecules, and hence the allergenicity, of a vespid venom protein.

[0031] Thus, there is a need in the art to determine how modification of B cell epitopes in the native structure of allergen proteins permits the design of improved therapeutics.

[0032] There is also a need in the art to provide allergen proteins that stimulate a B cell-mediated immune response without stimulating IgE mediated allergic responses. In particular, there is need in the art for providing allergens with a reduced density of epitopes that are efficient in stimulating an IgG production in B cells but are inefficient at crosslinking IgE antibodies specific for the native allergen bound to the surface of, for example and without limitation, mast cells or basophils.

[0033] There is also a need in the art to provide hybrid proteins bearing non-cross-reactive B cell epitopes that are effective in immunotherapy. In particular there is a need to for hybrid proteins that present allergen peptide epitope sequences in a conformation that is accessible to receptors on the surface of immune cells and soluble proteins, especially antibodies.

[0034] Hence, what are needed are agents, pharmaceutical compositions and methods for generating an IgG B cell response that provides protection against allergens, without eliciting an allergic reaction such as anaphylactic shock.

[0035] The citation of references herein shall not be construed as an admission that such is prior art to the present invention.

SUMMARY OF THE INVENTION

[0036] The present invention provides a new approach to prepare modified allergens. The modified allergens are hybrids consisting of a small portion of the “guest” allergen of interest and a large portion of a homologous but poorly cross-reacting “host” protein. The homologous host protein functions as a scaffold to maintain the native structure of the guest allergen of interest so that the conformation-dependent B cell epitopes of the guest allergen of interest are preserved in the hybrid, but at a reduced density. Homologous proteins of greater than 30% sequence identity and of similar functions are known to have closely similar three-dimensional structures (Chothia et al., 1990, Annual Review Biochem 59:1007; Russell, 1994, J Mol Biol 244:332), thus providing a plethora of guest/host proteins.

[0037] Thus, the present invention is directed to recombinant allergens, e.g., vespid venom allergens, of reduced allergenicity but that retain immunogenicity. Hence, the invention provides allergen protein, peptide epitope sequences corresponding to surface-accessible portions of the allergen, hybrid proteins comprising the peptide epitope sequences inserted in the corresponding structural region of the host scaffold, nucleic acids encoding such hybrid constructs, and methods that may be used to stimulate a therapeutic immune response to the allergens with reduced allergic response, i.e., an allergy immunotherapy. In particular, the recombinant hybrid proteins, nucleic acids and methods of the invention provide for stimulating a B cell-based response against the allergen, without triggering an IgE-based allergic response such as acute anaphylaxis.

[0038] The hybrid proteins of the present invention are present in a native conformation. In one embodiment hybrid proteins comprise at least one allergen peptide epitope sequence in a native conformation. More specifically, the scaffold protein and the native protein from which the allergen peptide epitope sequence is derived have the same native conformation.

[0039] In certain embodiments the hybrid proteins of the invention comprise a fusion peptide, such as a signal peptide or handle for purification. In other embodiments the hybrid proteins of the invention may comprise a protease processing site, e.g., for cleavage of the purification handle. Accordingly, the hybrid proteins of the invention comprises an allergen peptide epitope sequence, a scaffold protein sequence, and, optionally, either separately or in combination, a fused sequence and protease processing site.

[0040] The recombinant peptide epitope sequences are found on the surface of the native protein from which the sequence is derived. In a specific embodiment, the allergen peptide is a loop region of the native protein.

[0041] It will be appreciated that hybrid proteins may comprise more than one peptide epitope sequence introduced into the scaffold protein sequence.

[0042] The present invention extends to hybrid proteins wherein the peptide antigen is from a allergen protein and the scaffold protein is a heterologous protein having greater than or equal to 30% sequence identity to the native allergen protein. In a specific aspect, each of the peptide antigen and the scaffold protein are derived from vespid venom proteins. More specifically, the peptide antigen and scaffold proteins may be derived from vespid venom Ag 5s.

[0043] In one embodiment, the peptide epitope sequences of the present invention are characterized by having between about 6 and 50 amino acids and being antigenic in a mouse for a B cell response (B cell epitopes). More particularly, in examples of the invention, an allergen peptide epitope sequence of the invention is derived from an Ag peptide selected from the group consisting of:

[0044] NNYCKIKC (SEQ ID: 1);

[0045] NNYCKIKCLKGGVHTACK (SEQ II): 2);

[0046] NNYCKIKCLKGGVHTACKYGSLKP (SEQ ID: 3);

[0047] NNYCKIKCLKGGVHTACKYGSLKPNCGNKVVV (SEQ ID: 4);

[0048] NNYCKIKCLKGGVHTACKYGSLKPNCGNKVVVSYGLTKQ (SEQ ID: 5);

[0049] NNYCKIKCLKGGVHTACKYGSLKPNCGNKVVVSYGLTKQEKQDILK (SEQ ID: 6);

[0050] QVGQNVALTGSTAAKYDDPVKLVKMWEDEVKDYNPKKKFSGNDFL KTG (SEQ ID NO: 7);

[0051] HYTQMVWANTKEVGCGSIKYIQEKWHKHYLVCNYGPSGNFKNEELY QTK (SEQ ID NO: 8)

[0052] LKPNCGNKVVV (SEQ ID NO: 9);

[0053] LTGSTAAKYDD (SEQ ID NO: 10);

[0054] PKKKFSGND (SEQ ID NO: 11)

[0055] IQEKWHK (SEQ ID NO: 12); and

[0056] FKNEELYQTK (SEQ ID NO: 13);

[0057] NNYCKIKCLKGGVHTACKYGSLKPNCGNKVVVSYGLTKQEKQDILK EHND (SEQ ID NO: 93);

[0058] NNYCKIKCLKGGVHTACKYGSLKPNCGNKVVVSYGLTKQEKQDILK EHNDFRQKIAR (SEQ ID NO: 94);

[0059] NNYCKIKCLKGGVHTACKYGSLKPNCGNKVVVSYGLTKQEKQDILK EHNDFRQKIARGLETRGNPGPQPPAKNMKN (SEQ ID NO: 95).

[0060] The present invention further extends to an isolated expression vector comprising a promoter operationally associated with a nucleic acid of the invention. Numerous promoters commercially available to the skilled artisan can be used in this aspect of the invention. Examples include, but are not limited to immediate early promoters of hCMV, early promoters of SV40, early promoters of adenovirus, early promoters of vaccinia, early promoters of polyoma, late promoters of SV40, late promoters of adenovirus, late promoters of vaccinia, late promoters of polyoma, the lac the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or promoters of yeast α mating factor, to name only a few. Numerous examples of expression vectors having applications herein, and which are also readily available to the skilled artisan are described infra.

[0061] The invention also provides a method for preparing a nucleic acid that encodes an allergen hybrid protein of the invention. This method comprises introducing a nucleotide sequence encoding a peptide epitope sequence of an allergen protein into a nucleotide sequence encoding a scaffold protein that is structurally homologous to the allergen protein. The nucleotide sequence encoding the peptide epitope sequence is introduced in-frame with the nucleotide sequence encoding the scaffold protein, and in a location such that in the allergen hybrid protein the peptide epitope sequence is present in a surface accessible region of the hybrid protein corresponding to its position in the allergen protein. In one such embodiment, the nucleotide sequence encoding the scaffold protein is mutated to introduce the nucleotide sequence encoding the peptide epitope sequence. In another such embodiment, the nucleotide encoding the peptide epitope sequence is introduced by ligating fragments from nucleic acids comprising the nucleotide sequence encoding the peptide epitope sequence and the nucleotide sequence encoding the scaffold protein treated with an endonuclease. If necessary, endonuclease restriction sites can be introduced into the nucleic acids comprising such sequences using standard techniques in the art.

[0062] The present invention further extends to a method for producing a hybrid protein of the invention by expression of an isolated nucleic acid molecule of the invention. Such production provides a plentiful source of the hybrid protein for diagnosis and therapy. An example of such a method of the invention for producing a hybrid protein culturing a host cell transformed or transfected with an expression vector of the invention so that the host cell produces the hybrid protein of the invention. Preferably, the hybrid protein of the invention so produced from the culture, the host cell, or both is recovered.

[0063] The present invention further extends to pharmaceutical compositions effective for the treatment of an allergen-specific allergic condition. In particular, the present invention extends to a pharmaceutical composition comprising a hybrid protein of the invention, or a nucleic acid preferably an expression vector, encoding such a hybrid protein, and a pharmaceutically acceptable carrier thereof. The invention further includes pharmaceutical compositions containing a plurality of hybrid proteins of the invention, or containing a nucleic acid or nucleic acids encoding such a plurality.

[0064] Naturally, the present invention extends to a method for treating allergen-specific allergic condition comprising administering a therapeutically effective amount of a pharmaceutical composition of the invention. Administration of a pharmaceutical composition of the invention can occur by any route, and particularly orally, pulmonarily, nasally, topically or parenterally. Other routes of administration are also possible.

[0065] Yet another specific object of the invention is to provide a method for treating an allergen-specific allergy in a subject, wherein a pharmaceutical composition for treating an allergen-specific allergic condition is administered to the subject.

[0066] Moreover, the present invention extends to a pharmaceutical composition for modulating immune response of a mammal towards an immunogen, wherein the pharmaceutical composition comprises an allergen hybrid protein (or nucleic acid encoding such a protein) of the invention for modulating immune response towards an immunogen in a mammal, as set forth above, and a pharmaceutically acceptable carrier thereof.

[0067] As a result, administration of such a pharmaceutical composition modulates the immune system's ability to recognize and attack the immunogen. In a particular embodiment, the ability of the immune system of the mammal to recognize and attack the immunogen is increased upon administration of the pharmaceutical composition relative to the ability of the subject's immune system to recognize and attack the immunogen prior to administration of a pharmaceutical composition of the invention.

1ABBREVIATIONSDol mDolichovespula maculatawhite faced hornetDol aD. arenariayellow hornetPol aPolistes annulariswaspPol eP. exclamanswaspVes mVespula maculifronsyellowjacketVes vV. vulgarisyellowjacketPCRpolymerase chain reactionRACErapid amplification of cDNA endsTCRT cell receptor for antigen

BRIEF DESCRIPTION OF THE DRAWINGS

[0068]
FIG. 1. Ves v 5 cDNA [SEQ ID NO: 14] and amino acid [SEQ ID NO: 16] sequences. Numbering at L refers to nucleotide position; numbering at R refers to amino acid position.

[0069]
FIG. 2. Pol a 5 cDNA [SEQ ID NO: 15] and amino acid [SEQ ID NO: 17] sequence. Numbering at L refers to nucleotide position; numbering at R refers to amino acid position.

[0070]
FIG. 3. Amino acid comparison of Ves v 5 (V) [SEQ ID NO: 16] and Pol a 5 (P) [SEQ ID NO: 17].

[0071]
FIG. 4. Schematic sequence representations of Ag 5s and hybrids. Residue numbers given for hybrids refer to those of Ves v 5.

[0072]
FIG. 5A-B. Alignment of Ves v 5 homologous proteins from insect venoms from Vespula maculifrons [Ves m 5, SEQ ID NO: 63]; Vespula vulgaris [Ves v 5, SEQ ID NO: 64]; Vespula flavopilosa [Ves f 5, SEQ ID NO: 65]; Vespula pensylvanica [Ves p 5, SEQ ID NO: 66]; Vespula germanica [Ves g 5, SEQ ID NO: 67]; Vespula vidua [Ves vi 5, SEQ ID NO: 68]; Vespula squamosa [Ves s 5, SEQ ID NO: 69]; Dolichovespula maculata [Dol m 5a, SEQ ID NO: 70]; Dolichovespula arenaria [Dol a 5, SEQ ID NO: 71]; Dolichovespula maculata [Dol m 5b, SEQ ID NO: 72]; Vespa mandarinia [Vesp m 5, SEQ ID NO: 73]; Vespa crabro [Ves c 5.01, SEQ ID NO: 74]; Vespa crabro [Ves c 5.02, SEQ ID NO: 75]; Polistes fuscatus [Pol f 5, SEQ ID NO: 76]; Polistes exclamans [Pol e 5, SEQ ID NO: 77]; Polistes annularis [Pol a 5, SEQ ID NO: 78]; Solenopsis invicta [Sol i 3, SEQ ID NO: 79]; and Solenopsis richteri [Sol r 3, SEQ ID NO: 80].

[0073]
FIG. 6A-B. SDS gel patterns of Ag 5s and hybrids.

[0074]
FIG. 7. Circular dichroism (CD) spectra of Ves v 5 and hybrids.

[0075]
FIG. 8A-C. Inhibition ELISA with mouse antibodies specific for natural Ves v 5 using (A) Ves v 5-specific antibodies isolated from BALB/c mice and depleted of Pol a-cross reactive antibodies (B) antisera from ASW/n mice and (C) antisera from P/J mice.

[0076]
FIG. 9A-C. Inhibition ELISA with sera from yellow jacket-sensitive patients.

[0077]
FIG. 10 A-C. Binding of mouse Ves v 5-specific monoclonal antibodies to solid-phase Ves v 5 or hybrids.

[0078]
FIG. 11 A-C. Histamine release assay of Ves v 5, Pol a 5 and hybrids.

[0079]
FIG. 12A-B. Alignment of Ves v 5-like proteins. Aligned proteins are Ves v 5 [SEQ ID NO: 81]; Sol i 3 [SEQ ID NO: 82]; Lycopersicon esculentum p14a [SEQ ID NO: 83]; Schizophyllum commune SC7 [SEQ ID NO: 84]; human trypsin inhibitor [SEQ ID NO: 85]; human glipr [SEQ ID NO: 86]; Heloderma horridum helothermine [SEQ ID NO: 87]; and human TPX-1 [SEQ ID NO: 88].

DETAILED DESCRIPTION

[0080] The present invention is directed to recombinant allergen hybrid protein constructs of reduced allergenicity and but retaining immunogenicity, the nucleic acid molecules encoding such allergens, and methods of use for such allergens in the diagnosis and therapy of allergy. The hybrid proteins of the invention comprise a surface, e.g., loop or corner region, peptide epitope sequence introduced into a scaffold protein sequence. The hybrid proteins, nucleic acids and methods of the invention provide for stimulating a B cell-based response against the allergen without triggering an IgE-based allergic response. In a specific embodiment, a recombinant hybrid protein comprises a vespid venom surface or loop peptide antigen, particularly from Ves v 5, fused to a scaffold protein, particularly Pol a 5.

[0081] The invention is further directed to expression vectors comprising nucleic acid molecules that include allergen hybrid proteins of decreased allergenicity that retain immunogenicity, and to methods for producing such hybrid proteins of the invention by expressing and recovering such hybrid proteins.

[0082] The invention also provides pharmaceutical compositions effective for the treatment of an allergen-specific allergic condition comprising a hybrid protein of the invention or nucleic acid vector encoding such a hybrid protein, and methods for treating such allergic conditions comprising administering a therapeutically effective amount of such pharmaceutical compositions.

[0083] The hybrid proteins of the invention can also be useful for diagnosis of allergen-specific allergic conditions.

[0084] The present invention is based, in part, on the discovery that insertion of sequences from surface accessible regions of yellowjacket (Vespula vulgaris) antigen 5 into the corresponding region of Polistes annularis antigen 5 yielded a hybrid construct that retained the immunogenicity of the parent proteins, but showed significantly reduced allergenicity. Moreover, the most advantageous positions for introducing sequences were at surface accessible sites, especially loop and corner regions, as determined from the crystal structure of Ves v 5.

[0085] Earlier work established that hybrid constructs, in which one-quarter to one-third of the allergenic protein was introduced into the corresponding region of a homologous scaffold protein. However, these hybrid constructs lack the advantages and refinements of the present invention.

[0086] Clinical studies in patients and tests with experimental animals have shown that there is limited cross reactivity of antibodies specific for the yellow jacket and paper wasp venom proteins (Lichtenstein et al., 1979, J Allergy Clin Immunol 64:5; Lu et al., 1993, J Immunol 150:2823). These observations form the basis of a preferred embodiment of the present invention. A preferred guest allergen antigen 5 is Ves v 5, a yellow jacket venom protein of 23 kd. A preferred homologous host allergen, which serves as a scaffold protein, is Pol a 5, a paper wasp venom protein of similar size. Ves v 5 and Pol a 5 have 59% sequence identity (FIG. 3). Both can be expressed in yeast and the recombinant proteins were shown to have the native conformation of the natural proteins (Monsalve et al., 1999, Protein Expr. Purif. 16:410).

[0087] Immunochemical findings are reported for hybrids of Ves v 5 and Pol a 5. The sequence representations of these hybrids are shown schematically in FIG. 4. Hybrids PV1-46, PV109-155 and PV156-204 contain respectively the first one-quarter (i.e., amino acids 1-46), the third one-quarter (i.e., amino acids 109-155) and the last one-quarter (i.e., amino acids 156-204) of the Ves v 5 molecule, together with portions of the Pol a 5 molecule to complete the hybrid Ag 5 molecule. A hybrid containing the second one-quarter of the Ves v 5 molecule was not prepared, as this is a region of high sequence identity of Ves v 5 and Pol a 5 (see FIG. 3). Hybrid PV1-155 has the opposite arrangement of the Ves v 5 and Pol a 5 amino-terminal and carboxy-terminal fragments, when compared to PV156-204.

[0088] Hybrids PV1-8, PV1-18, PV1-24, PV1-32, PV22-32, PV115-125, PV142-150, PV176-182 and PV195-204 were designed to contain the surface, loop or corner regions of Ves v 5. These hybrids include 7-32 amino acids of Ves v Ag 5 substituted for a homologous region of Pol a Ag 5.

[0089] Switching corresponding regions of homologous proteins, especially in surface accessible, e.g., loop and corner, regions predictably conserves native structure. Surface accessible regions especially loop and corner regions, tend to demonstrate more flexibility and better tolerate changes while retaining structure. This approach also finds a counterpart in directed evolution, where homologous enzymes are recombined to yield novel, functional enzyme chimeras.

[0090] The term “allergen hybrid protein” refers to a recombinant or synthetic protein that has the native structure of the scaffold protein, but includes one or more sequences from an allergen. The allergen is a structural homolog of the scaffold protein, thus permitting introduction of the allergen sequences into corresponding positions in the scaffold protein. A “corresponding position” is the same position in the primary sequence or same topological position in the native structure. The allergen sequences are selected from a surface accessible region of the allergen and inserted in the corresponding surface accessible region of the scaffold protein. Because B cell epitopes of proteins in their native conformation are surface accessible, the sequences from the allergen introduced into the scaffold protein can act as B cell epitopes, hence they are called “peptide epitope sequences” of an allergen protein.

[0091] In connection with the present invention the expression “reduced allergenicity” means a molecule or antigen exhibits significantly reduced allergenic activity in an in vitro assay designed to measure such allergenicity. Such in vitro assays are well known in the art and include, for example and without limitation, assay of histamine release from basophils of a allergen sensitive patient or experimental animal following challenge. Furthermore, “activity” as used herein may refer to any measurable parameter or result that is indicative of the allergenicity of a molecule or antigen, such as, for example and without limitation, the maximum response obtained in an assay or the amount or concentration of antigen required to elicit a defined result in an assay.

[0092] The term “retaining immunogenicity” (in any grammatical form) means that the hybrid protein elicits an immune response, particularly an IgG-predominated humoral immune response, that is comparable to the immune response elicited by the native allergen or scaffold protein (or both) and greater than the allergic (IgE) immune response they elicit. The hybrid-specific IgG will cross react with epitopes present on the allergen and the scaffold protein. This IgG response can block IgE binding, thus reducing or preventing allergic responses. In addition, the hybrid protein may elicit T cell anergy and other allergy suppressive immune responses.

[0093] In accordance with the present invention, proteins are “homologous” if, following alignment, they exhibit at least about 30 percent amino acid identity, as determined by programs that are well know in the art, including, as non-limiting examples, the programs Gap, Bestfit and BLAST. More preferable is where homologous proteins exhibit at least 50 percent amino acid identity. However, in a specific embodiment the allergen protein and the scaffold protein do not have more than 70% sequence identity to reduce the possibility of a high degree of cross reactivity that might lead to an unaccepatable degree of allergenicity of the hybrid protein. Greater sequence identity can be tolerated, particularly where the peptide epitope sequence inserted in the scaffold protein is very dissimilar, e.g., less than 50% identical and preferably less than 30% identical, to the corresponding sequence from the scaffold protein that it replaces.

[0094] Proteins are structurally homologous when, due to primary sequence similarity, they adopt a similar core secondary and tertiary structure so that their three-dimensional structures can be superimposed with almost complete (greater than 70%) overlap. Their surface tertiary structure, however, may vary.

[0095] In a preferred embodiment of the present invention, peptide epitope sequences from the allergen are inserted into or replace sequences within “scaffold” proteins. Accordingly, a “scaffold protein” of the present invention is a protein which includes an allergen epitope sequence, either as an inserted sequence or as a replacement sequence for a homologous (corresponding) sequence of the scaffold protein. The scaffold protein adopts a native conformation. The allergen and scaffold can alternate positions; these terms are used to indicate the source of sequences (from the “allergen”) introduced into the “scaffold”. Because the “allergen” and “scaffold” are homologous, they are both likely to act as allergens, albeit to different populations. Thus, a “scaffold” can be an “allergen” if its surface accessible sequences are introduced into another structurally homologous protein.

[0096] The expression “native conformation” includes a functional conformation adopted by a non-recombinant, i.e., natural protein, polypeptide, or antigen, within its natural environment or following purification under conditions that maintain the functional conformation adopted in said natural environment. Native conformation can be measured, for example and without limitation, by determining the CD spectrum of a protein. Native conformation may also be determined by measuring enzymatic activity. It will be understood by the skilled artisan that, in cases where the functional conformation of a natural non-recombinant protein is unknown, “native conformation” will encompass forms of recombinant proteins that reproducibly exhibit a non-random defined conformation that includes secondary elements as typically found in properly folded functional proteins, such as for example, and without limitation, α helix and β sheet elements. It is also well known that, using recombinant techniques, additional amino acids may be joined to the amino or carboxyl end of a protein without disrupting the native conformation of the protein. Such additional amino acids may be short polypeptide “tags”, which are typically 1-25 amino acids in length and which are typically disordered, or longer polypeptides which may form a distinct domain, which may itself be ordered or disordered.

[0097] The expression “surface-exposed amino acid” means that an amino acid residue is located at the surface of the three-dimensional structure in such a manner that when the allergen is in solution at least a part of at least one atom of the amino acid residue is accessible for contact with the surrounding solvent. Preferably, the amino acid residue in the three-dimensional structure has a solvent (water) accessibility of at least 20%, more preferably at least 30%, still more preferably at least 40% and most preferably at least 50%.

[0098] The expression “solvent accessibility” is defined as the area of the molecule accessible to a sphere with a radius comparable to a solvent (water, r=1.4 Å) molecule. An “allergen” has its ordinary meaning, i.e., is any proteinacious molecule that elicits an allergic response, e.g., histamine release to anaphylactic shock. Allergens are well known; a representative group are listed in Table 8 of this specification. Examples of allergens according to the invention may suitably be an inhalation allergen originating, e.g., from trees, grasses, herbs, fungi, house dust mites, cockroaches and animal hair and dandruff. Important pollen allergens from trees, grasses and herbs are such originating from the taxonomic orders of Fagales, Oleales and Pinales including birch (Betula), alder (Alnus), hazel (Corylus), hombearn (Carpinus) and olive (Olea), the order of Poales including i.a. grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis and Secale, the orders of Asterales and Urticales including herbs of the generaAmbrosia and Artemisia. Important inhalation allergens from fungi are such originating from the genera Alternaria and Cladosporium. Other important inhalation allergens are those from house dust mites of the genus Dermatophagoides, those from cockroaches and those from mammals such as cat, dog and horse. Further, recombinant allergens according to the invention maybe mutants of venom allergens including such originating from stinging or biting insects such as those from the taxonomic order of Hymenoptera including bees (superfamily Apidae), wasps (superfamily Vespidea), and ants (superfamily Formicoidae). Specific allergen components include, e.g., Bet v 1 (B. verrucosa, birch), A/n g 1 (Alnus glutinosa, alder), Cor a 1 (Corylus avelana, hazel) and Car b 1 (Carpinus betulus, hornbeam) of the Fagales order. Others are Cryj 1 (Pinales), Amb a 1 and 2, Art v 1 (Asterales), Parj 1 (Urticales), Ole e 1 (Oleales), Ave e 1, Cyn d 1, Dac g 1, Fes p 1, Hol l 1, Lol p 1 and 5, Pas n 1, Phl p 1 and 5, Poa p 1, 2 and 5, Sec c 1 and 5, and Sor h 1 (various grass pollens), Alt a 1 and Cla h 1 (fungi), Der f 1 and 2, Der p 1 and 2 (house dust mites, D. farinae and D. pteronyssinus, respectively), Lep d 1 and 2 (Lepidoglyphus destructor; storage mite), Bla g 1 and 2, Per a 1 (cockroaches, Blatella germanica and Periplaneta americana, respectively), Fel d 1 (cat), Can f 1 (dog), Equ c 1, 2 and 3 (horse), Apis m 1 and 2 (honeybee), Ves v 1, 2 and 5, Pol a 1, 2 and 5 (all wasps) and Sol i 1, 2, 3 and 4 (fire ant). The term also includes all examples described in the “Background”, supra.

[0099] For example, the term “vespid venom allergen” refers to a protein found in the venom of a vespid, to which susceptible people are sensitized on exposure to the sting of the insect. While most antigens are characterized by being reactive with specific IgG class antibodies, an allergen is characterized by also being reactive with IgE type antibodies. The IgE type antibodies are responsible for mediating the symptoms of an allergic condition, i.e., immediate-type hypersensitivity.

[0100] As used herein, the term “vespid” is used according to the practice of those in the field of allergy, and refers to insects belonging to the worldwide family of Vespidae, i.e., social wasps including hornets, yellowjackets, and paper wasps. In particular, vespids include the subfamilies Vespinae and Polistinae. More particularly, the vespids include the genera Vespa Linnaeus, Vespula Thomson, Dolichovespula Rohwer, and Polistes Latreille. Species in the genus Vespula include but are not limited to V. germanica (Fab.), V. squamosa (Drury), V. maculifrons (Buysson), V. flavopilosa (Jacobson), V. vulgaris (L.), and V. pensylvanica (Saussure). Species in the genus Polistes include but are not limited to P. annularis (Linnaeus), P. exclamans (Viereck), P. metricus (Say), P. fuscatus (Fabricius), and P. apachus (Saussure). Species in the genus Dolichovespula include but are not limited to D. maculata (L.) and D. arenaria (Fab.). Species in the genus Vespa include but are not limited to V. crabro (L.) and V. orientalis (Linnaeus).

[0101] The taxonomic classification of Vespula vulgaris is as follows:

2OrderHymenopteraSuborderApocritaDivisionAculeataSuperfamilyVespoideaFamilyVespidaeSubfamilyVespinaeGenusVespulaSpecies GroupVespula vulgaris species groupSpeciesvulgaris

[0102] The taxonomic classification for Polistes annularis is as follows:

3OrderHymenopteraSuborderApocritaDivisionAculeataSuperfamilyVespoideaFamilyVespidaeSubfamilyPolistinaeTribePolistiniGenusPolistesSubgenusAphanilopterusSpeciesannularis

[0103] As used herein, the term “immunomodulatory” refers to an ability to increase or decrease an antigen-specific immune response, either at the B cell or T cell level. Immunomodulatory activity can be detected, e.g., in T cell proliferation assays, by measurement of antibody production, lymphokine production or T cell responsiveness. In particular, in addition to affects on B cell responses, the immunomodulatory polypeptides of the invention may bind to molecules on the surface of T cells, and affect T cell responses as well.

[0104] As used herein, the phrase “immune system related disease or disorder” refers to a disease or disorder that evokes an immune response in a subject, or effects the ability of the immune system to respond to an immunogen. Hence, examples of immune system related diseases or disorders comprise a pathogenic disease or disorder; a viral disease or disorder, e.g., HIV, Herpes Simplex virus, or papilloma virus; an autoimmune disease, e.g., arthritis or Lupus.

[0105] Determining Allergen Structure

[0106] The three-dimensional structure of a protein may be determined by physical methods that are well known in the art, including and without limitation, x-ray crystallography, nmr spectroscopy and electron crystallography. Preferred, the three-dimensional structure of a protein is determined by x-ray crystallography. It is also preferred that such techniques yield a resolution of 5 Å or better, at which resolution a trace of the α-carbons in the polypeptide backbone of a protein may be obtained, allowing the determination of protein secondary structure features, as for example, α-helix and β-sheet elements. More preferred is where the three dimensional structure of protein is determined at a resolution of 2 Å or better, at which resolution the position of amino acid side chains may be ascertained. Structures of specific allergens are well known, as set forth in Table 9. These, or others, can be determined using the standard techniques set forth above.

[0107] The three dimensional structure of a protein may also be inferred by comparison to an homologous protein, whose structure has been determined empirically by a physical method, as for example by aligning and comparing amino acid sequences. Methods for comparing and aligning amino acid sequences are well known in the art and include, for example and without limitation, the Pileup, Gap, BestFit and Compare programs (Genetic Computer Group, Madison, Wis.). Such alignment and comparison allows the identification of regions of high amino acid identity or similarity, which may adopt similar or identical conformations in homologous proteins. In this manner, once the three dimensional structure is determined for one protein, the three-dimensional structure may be determined for many homologous proteins, which allows for the identification of surface and loop regions of homologous proteins.

[0108] The three dimensional structure and function of a proteins is typically effected to a lesser extent by changes in amino acids located in surface and loop regions of proteins, compared to effects observed due to changes in internally located amino acids. The amino acid residues of surface and loop regions are therefore typically less conserved among homologous proteins, compared to internal residues. It will be appreciated by one of ordinary skill in the art, however, that surface and loop regions will occupy the same relative position in the native conformation of homologous proteins. The surface and loop regions therefore represent “conserved elements” or “homologous elements” within homologous proteins.

[0109] In addition, various spectroscopic techniques can be used to evaluate structure, particularly to confirm that the hybrid protein retains the native structure of the allergen and scaffold proteins. These techniques include, without limitation, circular dichroism spectroscopy, nmr spectroscopy (particularly at lower resolution), neutron diffraction, fluorescence spectroscopy (and other light absorption and transmission spectroscopic techniques), and the like. In particularly, evaluating identity of spectra can indicate the degree to which the hybrid protein adopts the native conformation. Circular dichroism spectroscopy provides a preferred tool for this type of evaluation.

[0110] Molecular Biological Techniques

[0111] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, “Molecular Cloning: a Laboratory Manual,” Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); “DNA Cloning: a Practical Approach,” Volumes I and II (D. N. Glover ed. 1985); “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription And Translation” [B. D. Hames & S. J. Higgins, eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning” (1984). Other techniques in accordance with the present invention may be found in U.S. Pat. Nos. 5,593,877; 5,612,209, 5,804,201, 6,106,844 and U.S. application Ser. Nos. 08/484,388, 08/474,853, and 09/166,205 to King and in Monsalve et al. (1999, Protein Expr. Purif. 16:410).

[0112] A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”) in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules, restriction fragments, viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.

[0113] A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. For preliminary screening for homologous nucleic acid molecules, low stringency hybridization conditions, corresponding to a Tm of 55°, can be used, e.g., 5× SSC, 0.1% SDS, 0.25% non-fat dry milk, and no formamide; or 30% formamide, 5× SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5× or 6× SSC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5× or 6× SSC. Hybridization requires that the two nucleic acid molecules contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acid molecules depends on the length of the nucleic acid molecules and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acid molecules having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-0.51). For hybridization with shorter nucleic acid molecules, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). Preferably a minimum length for a hybridizable nucleic acid molecule is at least about 10 nucleotide; more preferably the length is at least about 20 nucleotides; even more preferably at least about 30 nucleotides; and most preferably at least about 40 nucleotides.

[0114] In a specific embodiment, the term “standard hybridization conditions” refers to a Tm of 55° C., and utilizes conditions as set forth above. In a preferred embodiment, the Tm is 60° C.; in a more preferred embodiment, the Tm is 65° C.

[0115] A DNA “coding sequence” or “encoding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

[0116] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

[0117] A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes.

[0118] A coding sequence is “under the contro” of or “operationally associated” with transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. A “signal sequence” can be included before the coding sequence. This sequence encodes a “signal peptide”, N-terminal to the polypeptide, that directs the host cell to transport the polypeptide to the cell surface or secrete the polypeptide into the media. The signal peptide is usually selectively degraded by the cell upon exportation. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

[0119] A “nucleic acid molecule” refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; “RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; “DNA molecules”) in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules, restriction fragments, viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A “recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.

[0120] A nucleic acid molecule is “hybridizable” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., supra). The conditions of temperature and ionic strength determine the “stringency” of the hybridization. For preliminary screening for homologous nucleic acid molecules, low stringency hybridization conditions, corresponding to a Tm of 55°, can be used, e.g., 5× SSC, 0.1% SDS, 0.25% non-fat dry milk, and no formamide; or 30% formamide, 5× SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5× or 6× SSC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5× or 6× SSC. Hybridization requires that the two nucleic acid molecules contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acid molecules depends on the length of the nucleic acid molecules and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acid molecules having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-0.51). For hybridization with shorter nucleic acid molecules, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). Preferably a minimum length for a hybridizable nucleic acid molecule is at least about 10 nucleotide; more preferably the length is at least about 20 nucleotides; even more preferably at least about 30 nucleotides; and most preferably at least about 40 nucleotides.

[0121] In a specific embodiment, the term “standard hybridization conditions” refers to a Tm of 55° C., and utilizes conditions as set forth above. In a preferred embodiment, the Tm is 60° C.; in a more preferred embodiment, the Tm is 65° C.

[0122] A DNA “coding sequence” or “encoding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

[0123] Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

[0124] A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes.

[0125] A coding sequence is “under the control” of or “operationally associated” with transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. A “signal sequence” can be included before the coding sequence. This sequence encodes a “signal peptide”, N-terminal to the polypeptide, that directs the host cell to transport the polypeptide to the cell surface or secrete the polypeptide into the media. The signal peptide is usually selectively degraded by the cell upon exportation. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

[0126] Nucleic Acid Molecules Encoding Hybrid Proteins

[0127] The invention relates to isolated nucleic acid molecules encoding recombinant allergen hybrid proteins. The invention further relates to a cell line stably containing a recombinant nucleic acid molecule encoding a allergen hybrid protein, and capable of expressing such nucleic acid molecule to produce the hybrid protein. The nucleic acids can be generated from allergens, e.g., as listed in Table 8 and in certain patents and patent applications disclosed herein.

[0128] As a specific example, the present disclosure provides the complete nucleic acid sequence of a vespid venom protein. In particular, the present disclosure provides the nucleic acid sequence of a vespid Ag 5, in particular Ves v Ag 5 (SEQ ID NO: 14; see FIG. 1) and Pol a Ag 5 (SEQ ID NO:15; see FIG. 2). Also provided are the amino acid sequences of Ves v Ag 5 (SEQ ID NO: 16; see FIG. 1) and Pol a Ag 5 (SEQ ID NO: 17; see FIG. 2).

[0129] In a specific embodiment, to obtain a nucleic acid molecule of the invention, DNA fragments are amplified by polymerase chain reaction (PCR) to amplify a fragment encoding a sequence comprising the allergen peptide epitope sequence or a scaffold protein. Oligonucleotide primers representing an allergen protein or scaffold protein of the invention can be used as primers in PCR. Generally, such primers are prepared synthetically. PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp™).

[0130] Nucleic acids of the invention may also be obtained by cloning of restrictions fragments. Alternatively, nucleic acids of the invention may be obtained by recombination of nucleic acids in vivo or in vitro. In some instances recombination depends on sequence homology between the nucleic acids that participate in a recombination event, but in other instances the nucleic acids undergoing recombination need not contain significant homology, as is the case, for example, in “illegitimate” recombination events. One of ordinary will recognize recombination of nucleic acids may be an inter- or intramolecular event.

[0131] Alternatives to isolating the allergen proteins or scaffold DNA or cDNA include, but are not limited to, chemically synthesizing the gene sequence itself from the sequence provided herein.

[0132] The above methods are not meant to limit the methods by which DNA of the invention may be obtained.

[0133] The methods used to obtain a nucleic acid of the invention may lead to the insertion or deletion of nucleotides at junctions where nucleic acids are joined, by recombinant or other techniques. In one embodiment, nucleotides may be inserted or deleted at the junction of a nucleic acid encoding an antigenic peptide and the nucleic acid encoding a scaffold protein. Such nucleic acids are fully within the scope of the invention. Accordingly, the invention encompasses hybrid proteins wherein amino acids have been inserted or deleted at the junction of a peptide epitope sequence and a scaffold protein sequence.

[0134] Nucleic acid sequence of the cloned hybrid protein, or starting materials thereof, can be modified by any of numerous strategies known in the art (Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the nucleic acid encoding a hybrid protein, care should be taken to ensure that the modified nucleic acid remains within the same translational reading frame as the scaffold protein, uninterrupted by translational stop signals.

[0135] Additionally, the nucleic encoding an allergen peptide epitope sequence or scaffold protein can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551; Zoller and Smith, 1984, DNA 3:479-488; Oliphant et al., 1986, Gene 44:177; Hutchinson et al., 1986, Proc. Natl. Acad. Sci. U.S.A. 83:710), use of TAB® linkers (Pharmacia), etc. PCR techniques are preferred for site directed mutagenesis (see Higuchi, 1989, “Using PCR to Engineer DNA”, in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

[0136] A large number of vector-host systems known in the art may be used to express a DNA of the invention. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as various pBR322 derivatives, for example, pUC, CR, pGEX vectors, pmal-c, pFLAG, etc. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. In a preferred aspect of the invention, the PCR amplified nucleic acid molecules of the invention contain 3′-overhanging A-nucleotides, and can be used directly for cloning into a pCR vector with compatible T-nucleotide overhangs (Invitrogen Corp., San Diego, Calif.). However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and a DNA of the invention may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.

[0137] In specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate the DNA of the invention enables generation of multiple copies of the DNA. Thus, the DNA may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted sequences from the isolated recombinant DNA.

[0138] The nucleotide sequences encoding Ves v 5 polypeptide epitope sequences of SEQ ID NO: 1-13 and 93-95 are given respectively in SEQ ID NO: 18-30 and 96-98.

[0139] Expression of an Allergen Hybrid Protein

[0140] The nucleotide sequence coding for a hybrid protein or an immunomodulatory fragment, derivative or analog thereof, can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such elements are termed herein a “promoter.” Thus, the nucleic acid molecule encoding the hybrid protein is operationally associated with the promoter. An expression vector also preferably includes a replication origin. The necessary transcriptional and translational signals can also be supplied by the native gene encoding the allergen or scaffold protein and/or its flanking regions. Potential host-vector systems include but are not limited to mammalian cell systems, e.g., infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems, e.g., infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

[0141] In an alternative embodiment, a recombinant hybrid protein of the invention, or an immunomodulatory fragment, derivative or analog thereof, is expressed chromosomally, after integration of the hybrid protein coding sequence by recombination. In this regard, any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook et al., 1989, supra, at Section 16.28).

[0142] The cell into which the recombinant vector comprising the nucleic acid molecule encoding the hybrid protein is cultured in an appropriate cell culture medium under conditions that provide for expression of the hybrid protein by the cell. The expressed hybrid protein can then be recovered from the culture according to methods well known in the art. Such methods are described in detail, infra.

[0143] In a another embodiment, a hybrid protein can be expressed initially with amino acids that are subsequently cleaved from the hybrid protein. The sequences to be removed can be amino- or carboxyl-terminal to the hybrid protein sequences. The sequences may be removed either in vivo or in vitro. Preferably the sequences are removed by cleavage at a specific site by a protease, e.g., signal peptidase, Factor Xa, Kex2 or a dipeptidyl amino peptidase. A recombinant DNA molecule encoding such a hybrid protein that includes a polypeptide to be cleaved by a protease comprises a sequence encoding the peptide to be cleaved from the hybrid protein joined in-frame to the coding sequence for a allergen hybrid.

[0144] In a specific embodiment, the hybrid proteins are expressed with an additional sequence comprising about six histidine residues, e.g., using a pQE vector (QIAGEN, Chatsworth, Calif.). The presence of the histidine makes possible the selective isolation of recombinant proteins on a Ni-chelation column. Other such handles include, but are not limited to, FLAG, a myc tag, GST, etc.

[0145] In another embodiment, a periplasmic form of the hybrid protein (containing a signal sequence) can be produced for export of the protein to a yeast periplasm or into a culture medium. Export to the periplasm or into the medium can promote proper folding of the expressed protein.

[0146] Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).

[0147] Expression of nucleic acid sequence encoding a hybrid protein, or an immunomodulatory fragment thereof, may be regulated by a second nucleic acid sequence so that the hybrid protein is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a hybrid protein may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression. Promoters which may be used to control expression of the hybrid protein coding sequences include, but are not limited to, the CMV promoter, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25); see also “Useful proteins from recombinant bacteria” in Scientific American, 1980, 242:74-94; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and the animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals.

[0148] In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage [e.g. of a signal sequence]) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an nonglycosylated core protein product. However, the enzyme protein expressed in bacteria may not be properly folded. Expression in yeast can produce a glycosylated product. Expression in insect cells can be used to increase the likelihood of native glycosylation and folding of a heterologous allergen hybrid protein. Furthermore, different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent.

[0149] Vectors are introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell hybrid, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed Mar. 15, 1990).

[0150] Both cDNA and genomic sequences can be cloned and expressed.

[0151] It is further contemplated that the hybrid proteins of the present invention, or fragments, derivatives or analogs thereof, can be prepared synthetically, e.g. by solid phase peptide synthesis.

[0152] Once the recombinant hybrid protein is identified, it may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, size exclusion, and reverse phase chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

[0153] In a particular embodiment, a hybrid protein and fragments thereof can be engineered to include about six histidyl residues, which makes possible the selective isolation of the recombinant protein on a Ni-chelation column. In a preferred aspect, the proteins are further purified by reverse phase chromatography.

[0154] In another embodiment, the recombinant hybrid protein may include additional sequences that allow the hybrid protein to be targeted for affinity purification such as FLAG, MYC, or GST (glutathione-S-transferase). For example, antibody specific for the additional sequences of the hybrid protein can be immobilized on a solid support, e.g., cyanogen bromide-activated Sepharose, and used to purify the hybrid protein. In another embodiment, a binding partner of the additional sequences, such as a receptor or ligand, can be immobilized and used to affinity purify the hybrid protein.

[0155] In one embodiment, the hybrid protein, preferably purified, is used without further modification, i.e., without cleaving or otherwise removing any sequences that maybe present in addition to the peptide epitope sequence and the scaffold protein. In a preferred embodiment, the hybrid protein can be used therapeutically, e.g., to modulate an immune response.

[0156] In a further embodiment, the purified hybrid protein is treated to cleave and remove any sequences that may have been added to the scaffold protein. For example, where the hybrid protein has been prepared to include a protease sensitive cleavage site, the hybrid protein can be treated with the protease to cleave the protease specific site and release the hybrid protein. In a specific embodiment, the hybrid protein is cleaved by treatment with Factor Xa.

[0157] In particular embodiments, recombinant hybrid proteins of the present invention include but certainly are not limited to those comprising, as a vespid venom antigen, a Ves v 5 peptide of SEQ ID NO: 1-13 or 93-95.

[0158] In a particular embodiment, recombinant vespid venom hybrid proteins of the present invention include but certainly are not limited to those comprising, as a scaffold protein, Pol a 5 protein of SEQ ID NO: 17.

[0159] Hybrid proteins can contain altered epitope or scaffold, or both, sequences, in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0160] Manipulations of the recombinant hybrid protein may also be made at the protein level such as glycosylation, acetylation, phosphorylation, amidation, reduction and carboxymethylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4; acetylation, formylation, oxidation, reduction; metabolic synthesis in the presence of tunicamycin; etc.

[0161] In a particular embodiment, the hybrid protein is expressed in an insect cell expression system, e.g., using a baculovirus expression vector. In a preferred embodiment, the hybrid protein is expressed in yeast, e.g., without limitation, Picchia pastoris, using appropriate expression systems. As pointed out above, these expression systems should yield “native” glycosylation and structure, particularly secondary and tertiary structure, of the expressed polypeptide.

[0162] Activity Assays With Hybrid Proteins of the Invention

[0163] Numerous assays are known in immunology for evaluating the immunomodulatory activity of an antigen. For example, the hybrid proteins can be tested for the ability to bind to antibodies specific for the allergen or the scaffold. Preferably, such antibodies that are detected in the diagnostic assay are of the IgG or IgE class. Hybrid proteins produced in eukaryotic expression systems, and particularly yeast cell expression systems, can have the correct structure for antibody binding. Hybrid proteins expressed in bacterial expression systems may not, and would thus require refolding prior to use in a diagnostic assay for antibody binding.

[0164] In another embodiment, the hybrid proteins of the invention can be tested in a proliferation assay for T cell responses. For such T cell response assays, the expression system used to produce the protein does not appear to affect the immunomodulatory activity of the protein. Generally, lymphocytes from a sensitized host are obtained. The host can be a mouse that has been immunized with an allergen, scaffold or hybrid protein, such as a vespid venom Ag 5 that has been produced recombinantly.

[0165] In a preferred embodiment, peripheral blood leukocytes are obtained from a human who is sensitive to the allergen. Using techniques that are well known in the art, T lymphocyte response to the protein can be measured in vitro. In a specific embodiment, infra, T cell responses are detected by measuring incorporation of 3H-thymidine, which increases with DNA synthesis associated with proliferation.

[0166] Cell proliferation can also be detected using an MTT assay (Mossman, 1983, J. Immunol. Methods 65:55; Niks and Otto, 1990, J. Immunol. Methods 130:140). Any method for detecting T cell proliferation known in the art can be used with the vespid protein produced according to the present invention.

[0167] Similarly, lymphokine production assays can be practiced according to the present invention. In one embodiment, lymphokine production can be assayed using immunological or co-stimulation assays (see, e.g., Fehlner et al., 1991, J. Immunol. 146:799) or using the ELISPOT technique (Czerkinsky et al., 1988, J. Immunol. Methods 110:29). Alternatively, mRNA for lymphokines can be detected, e.g., by amplification (see Brenner et al., 1989, BioTechniques 7:1096) or in situ hybridization (see, e.g., Kasaian and Biron, 1989, J. Immunol. 142:1287). Of particular interest are those individuals whose T cells produce lymphokines associated with IgE isotype switch events, e.g., IL-4 and IL-5 (Purkeson and Isakson, 1992, J. Exp. Med. 175:973).

[0168] Thus, in a preferred aspect, the hybrid proteins produced according to the present invention can be used in in vitro assays with peripheral blood lymphocytes or, more preferably, cell lines derived from peripheral blood lymphocytes, obtained from allergen sensitive individuals to detect secretion of lymphokines ordinarily associated with allergic responses, e.g., IL-4. Such assays may indicate which component or components of the hybrid protein are responsible for the allergic condition.

[0169] Therapeutic Uses of the Hybrid Protein and Nucleic Acid Vectors

[0170] The present invention provides a plentiful source of a hybrid protein, e.g., produced by recombinant techniques. Alternatively, a hybrid protein can be produced by peptide synthesis.

[0171] The invention contemplates use of hybrid proteins in therapeutic (pharmaceutical) compositions, for the use in the therapy of allergen-specific allergic conditions, treating allergen-specific allergic conditions, immune system related conditions, and modulating immune response in a mammal against an immunogen. In a specific embodiment, Ves v 5 and Pol a 5 hybrid proteins, or derivatives or analogs thereof, are contemplated for use in diagnosis, therapy, treatment, and modulation of immune response according to the present invention.

[0172] The phrase “therapeutically effective amount” is used herein to mean an amount sufficient to treat, and preferably increase by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, the ability of the immune system of a subject to combat effectively an immunogen. As further studies are conducted, information will emerge regarding appropriate dosage levels for modulation of immune system response towards an immunogen in various patients, and the ordinary skilled worker, considering the therapeutic context, age and general health of the recipient, will be able to ascertain proper dosing.

[0173] Therapeutic Methods

[0174] Therapeutic compositions of the invention (see, infra) can be used in immunotherapy, also referred to as hyposensitization therapy. Immunotherapy has proven effective in allergic diseases, particular insect allergy. Allergens are administered parenterally over a long period of time in gradually increasing doses. Such therapy may be particularly effective when the allergen or allergens to which the patient is sensitive have been specifically identified and the therapy is targeted to those allergen(s). However, this approach suffers the drawback of potentially precipitating an allergic reaction; especially anaphylaxis. Thus, the availability of hybrid proteins in large quantities is important for immunotherapy of allergy because they induce an effective IgG response against the allergen without an allergic reaction.

[0175] As discussed in the Background of the Invention, the presence of B cell epitopes on an allergen can cause an undesirable systemic reaction when the allergen is used for immunotherapy. Thus, a particular advantage of the invention is the capability to provide allergen polypeptides that do not cause undesirable systemic effects.

[0176] In one embodiment, one or more hybrid proteins can be injected subcutaneously to decrease the T cell response to the native molecule, e.g., as described by Brine et al. (1993, Proc. Natl. Acad. Sci. U.S.A. 90:7608-12).

[0177] In another embodiment, one or more hybrid proteins can be administered intranasally to suppress allergen-specific responses in naive and sensitized subjects (see e.g., Hoyne et al., 1993, J. Exp. Med. 178:1783-88).

[0178] Administration of a hybrid protein of the invention is expected to induce a strong anti-allergen B cell (antibody), IgG response that will block IgE antibodies, and thus, have a therapeutic effect.

[0179] These results can also be achieved by administration of a vector that permits expression of the hybrid protein, i.e., by gene therapy. Preferred vectors, particularly for cellular assays in vitro and in vivo, are viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia virus, baculovirus, alphaviruses (especially Sindbis viruses and Semliki Forest viruses), and other recombinant viruses with desirable cellular tropism; and non-viral vectors. For gene therapy in vivo or ex vivo, a pharmaceutically acceptable vector is preferred, such as a replication incompetent viral vector. Pharmaceutically acceptable vectors containing the nucleic acids of this invention can be further modified for transient or stable expression. As used herein, the term “pharmaceutically acceptable vector” includes, but is not limited to, a vector or delivery vehicle having the ability to selectively target and introduce the nucleic acid into cells.

[0180] Thus, a gene encoding a functional or mutant protein or polypeptide domain fragment thereof can be introduced in vivo, ex vivo, or in vitro using a viral vector or through direct introduction of DNA. Expression in targeted tissues can be affected by targeting the transgenic vector to specific cells, such as with a viral vector or a receptor ligand, or by using a tissue-specific promoter, or both. Targeted gene delivery is described in PCT Publication No. WO 95/28494.

[0181] Viral vectors commonly used for in vivo or ex vivo targeting and therapy procedures are DNA-based vectors and retroviral vectors. Methods for constructing and using viral vectors are known in the art (see, e.g., Miller and Rosman, BioTechniques 1992, 7:980-990). Preferably, the viral vectors are replication-defective, that is, they are unable to replicate autonomously in the target cell. Preferably, the replication defective virus is a minimal virus, i.e., it retains only the sequences of its genome that are necessary for encapsidating the genome to produce viral particles.

[0182] DNA viral vectors include an attenuated or defective DNA virus, such as but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), alphavirus (especially Sindbis virus), and the like. Defective viruses that entirely or almost entirely lack viral genes are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, a specific tissue can be specifically targeted. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 1991, 2:320-330), defective herpes virus vector lacking a glyco-protein L gene, or other defective herpes virus vectors (PCT Publication Nos. WO 94/21807 and WO 92/05263); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al. (J. Clin. Invest. 1992, 90:626-630; see also La Salle et al., Science 1993, 259:988-990); a defective adeno-associated virus vector (Samulski et al., J. Virol., 1987, 61:3096-3101; Samulski et al., J. Virol. 1989, 63:3822-3828; Lebkowski et al., Mol. Cell. Biol. 1988, 8:3988-3996); and Alphavirus vectors, including Sindbis virus and Semliki Forest virus-based vectors (U.S. Pat. No. 5,091,309; PCT Publication No. WO 98/44132; Schlesinger and Dubensky, Curr. Opin. Biotechnol. 1999, 5:434-9; Zaks et al., Nat. Med. 1999, 7:823-7).

[0183] Various companies produce viral vectors commercially, including, but not limited to, Avigen, Inc. (Alameda, Calif.; AAV vectors), Cell Genesys (Foster City, Calif.; retroviral, adenoviral, AAV, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, Pa.; adenoviral and AAV vectors), Genvec (France; adenoviral vectors), IntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and herpes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).

[0184] In another embodiment, the vector can be introduced in vivo by lipofection, as naked DNA, or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner, et. al., Proc. Natl. Acad. Sci. USA 1987, 84:7413-7417; Feigner and Ringold, Science 1989, 337:387-388; see Mackey, et al., Proc. Natl. Acad. Sci. USA 1988, 85:8027-8031; Ulmer et al., Science 1993, 259:1745-1748). Useful lipid compounds and compositions for transfer of nucleic acids are described in PCT Patent Publication Nos. WO 95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127. Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et. al., supra). Targeted peptides, e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.

[0185] Other molecules are also useful for facilitating transfection of a nucleic acid in vivo, such as a cationic oligopeptide (e.g., PCT Patent Publication No. WO 95/21931), peptides derived from DNA binding proteins (e.g., PCT Patent Publication No. WO 96/25508), or a cationic polymer (e.g., PCT Patent Publication No. WO 95/21931).

[0186] It is also possible to introduce the vector in vivo as a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see, e.g., Wu et al., J. Biol. Chem. 1992, 267:963-967; Wu and Wu, J. Biol. Chem. 1988, 263:14621-14624; Canadian Patent Application No. 2,012,311; Williams et al., Proc. Natl. Acad. Sci. USA 1991, 88:2726-2730). Receptor-mediated DNA delivery approaches can also be used (Curiel et al., Hum. Gene Ther. 1992, 3:147-154; Wu and Wu, J. Biol. Chem. 1987, 262:4429-4432). U.S. Pat. Nos. 5,580,859 and 5,589,466 disclose delivery of exogenous DNA sequences, free of transfection facilitating agents, in a mammal. Recently, a relatively low voltage, high efficiency in vivo DNA transfer technique, termed electrotransfer, has been described (Mir et al., C. P. Acad. Sci. 1988, 321:893; PCT Publication Nos. WO 99/01157, WO 99/01158, and WO 99/01175).

[0187] Treatment of Immune System Related Diseases

[0188] As explained above, the present invention relates to hybrid proteins for treating immune system related diseases or disorders, or for modulating immune response in a mammal towards an immunogen. In particular, Applicant has discovered that the hybrid proteins of the invention have applications in modulating a subject's immune response to various immunogens, in a manner that elicits an immune response without eliciting an allergenic response. In a particular embodiment, hybrid proteins of the invention modulate a subject's immune system to have increased ability to combat pathogens and viruses including, but not limited to, HIV, Herpes Simplex virus, or papilloma virus. Such a method comprises administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a polypeptide encoded by an isolated nucleic acid molecule comprising a DNA molecule of the invention. Furthermore, it has been discovered that the hybrid proteins, nucleic acids and vectors of the invention also have applications in treating an immune system related disease or disorder, or a symptom related thereto. As used herein, the phrase “immune system related disease or disorder” refers to a disease or disorder which evokes an immune response in a subject, or effects the ability of the immune system to respond to an immunogen. Examples of immune system related diseases or disorders which can be treated with agents and pharmaceutical compositions of the invention include, but are not limited to, a pathogenic disease or disorder; a viral disease or disorder, e.g. HIV, Herpes Simplex virus, or papilloma virus; or an autoimmune disease, e.g. arthritis or Lupus.

[0189] Moreover, the present invention extends to a method for treating an immune system related disease or disorder, or a symptom related thereto, comprising administering a therapeutically effective amount of a pharmaceutical composition for treating an immune system related disease or disorder to a subject. Hence, for example, should the immune system related disease or disorder involve HIV, a clinically significant change would, for example, involve an increase in white blood cell count in a subject to whom a pharmaceutical composition of the invention is administered relative to white blood cell count prior to administration. Other such examples of monitoring a clinically significant change in a subject will be readily apparent to one of ordinary skill in the art. Furthermore, as further studies are conducted, information will emerge regarding appropriate dosage levels for treating an immune system related disease or disorder, or a symptom related thereto in various patients, and the ordinary skilled worker, considering the therapeutic context, age and general health of the recipient, will be able to ascertain proper dosing. Examples of pharmaceutically acceptable compositions are described infra.

[0190] Pharmaceutically Acceptable Compositions

[0191] The in vivo therapeutic compositions of the invention may also contain appropriate pharmaceutically acceptable carriers, excipients, diluents and adjuvants. As used herein, the phrase “pharmaceutically acceptable” preferably means approved by a regulatory agency of a government, in particular the Federal government or a state government, or listed in the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animals, and more particularly in humans. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.

[0192] Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include mannitol, human serum albumin (HSA), starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0193] Such compositions will contain an effective diagnostic or therapeutic amount of the active compound together with a suitable amount of carrier so as to provide the form for proper administration to the patient. While intravenous injection is a very effective form of administration, other modes can be employed, such as by injection, or by oral, nasal or parenteral administration.

[0194] The invention will be further clarified by the following examples, which are intended to be purely exemplary of the invention.

EXAMPLE 1

Construction of Ag5 Hybrid cDNAs

[0195] Primers 1-24 used in the Examples are listed in Table 1.

[0196] Ves v 5 EA and KR constructs were prepared by PCR amplification of Ves v 5 cDNA template (Lu et al., 1993, J. Immunol. 150:2823) with the primers 1 (SEQ ID NO: 31) and 3 (SEQ ID NO: 33) or 2 (SEQ ID NO: 32) and 3 (SEQ ID NO: 33), respectively. Pol a 5 EA and KR constructs were prepared by PCR amplification of a Pol a cDNA template (Lu et al., 1993, J. Immunol. 150:2823) with the primers 4 (SEQ ID NO: 34) and 6 (SEQ ID NO: 24) or 5 (SEQ ID NO: 35) and 6 (SEQ ID NO: 36), respectively. Each cDNA construct contained an EcoRi or XhoI site at the 5′terminus and an XbaI site at the 3′-terminus. cDNAs were cloned in the plasmid vector pPICZαA (Invitrogen Corp, San Diego, Calif.) as either EcoRi -XbaI or XhoI-XbaI fragments. Positive clones were identified by PCR. The sequences of recombinant Ag5 and hybrid cDNAs in pPICZαA were confirmed by DNA sequencing of the inserts. Other constructs were prepared as described in King et al. (2001, J. Immunol. 166:6057-6065).

[0197] (i) PV1-46. The PV1-46 hybrid was constructed by joining amino-terminal sequences of Ves v 5 and carboxyl-terminal sequences of Pol a 5 at the peptide sequence EH, which is present at amino acids 47-48 and 49-50 of the respective proteins. The nucleotide sequence encoding the EH peptide in Ves v 5 is GAG CAC, which corresponds to a Bsi HKA I restriction enzyme cleavage site.

[0198] To facilitate construction of the PV1-46 hybrid, the natural DNA sequence (GAG CAT) encoding the Pol a 5 EH peptide at amino acids 49-50 was mutated to a Bsi HKA I site by a PCR overlap extension method (Ho et al., 1989, Gene 77:51), as follows. A first step comprised two separate PCRs. In one PCR, primers 4 (SEQ ID NO: 34) and 8 (SEQ ID NO: 38) and were used to amplify DNA encoding residues 1-53 of Pol a 5 wherein the EH-encoding sequence was converted to a Bsi HKA I site. In a second PCR, primers 7 (SEQ ID NO: 37) and 6 (SEQ ID NO:36) were used to amplify DNA encoding residues 47-205 of Pol a 5 wherein the EH-encoding sequence was converted to a Bsi HKA I site. Both PCRs were performed with 1-40 ng Pol a cDNA as template and 50 pmole each of sense and anti-sense primers in 100 μl of PCR buffer containing 0.2 mM dNTPs and 5 units Taq polymerase. Cycling conditions were 0.5 min denaturation at 95°, 0.5 min annealing at 55° and 2 min extension at 72° for 35 cycles. The products of these two PCRs contained an overlap region. In the second step of the overlap extension procedure, the purified products of the first two reactions were mixed to served as the template for a third PCR with flanking primers 4 (SEQ ID NO: 34) and 6 (SEQ ID NO: 36), yielding a full length Pol a 5 with the EH-encoding sequence converted to a Bsi HKA I site.

[0199] Hybrid PV1-46 encoding cDNA was then prepared by ligation of the appropriate Bsi HKA I fragments from Ves v 5 and the modified Pol a 5 cDNAs into pPICZαA, as described above for Ag5 encoding cDNAs.

[0200] (ii) PV109-155. The PV109-155 hybrid was constructed by joining amino-terminal sequences of Ves v 5 and carboxyl-terminal sequences of Pol a 5 at the peptide sequence KY, which is present at amino acids 106-107 and 109-110 of the respective proteins. The KY peptides of both Ag 5s are encoded by the nucleotide sequence AAA TAT. To construct PV109-155, KY-encoding sequences of appropriate Ag5 or hybrid cDNAs were mutated to an Apo I restriction enzyme cleavage site (AAA TTT) encoding a peptide sequence of KF. These single base mutations were made using the PCR overlap extension method (Ho et al., 1989, Gene 77:51) described in Example 1. In one set of reactions, the KY-encoding nucleotide sequence of PV1-155 cDNA was converted by performing the PCR overlap procedure with mutagenic primers 9 (SEQ ID NO: 39) and 10 (SEQ ID NO: 40). In a second set of reactions, the KY-encoding nucleotide sequence of Pol a 5 cDNA was converted by performing the PCR overlap procedure with mutagenic primers 11 (SEQ ID NO: 41) and 12 (SEQ ID NO: 42). Hybrid PV109-155 encoding cDNA was prepared by ligation of the appropriate fragments from Apo I digestions of converted Pol a 5 and converted PV1-155 encoding cDNAs into pPICZαA.

[0201] (iii) PV1-155 and PV156-204. Ves v 5 and Pol a 5 cDNAs have a common Eae I restriction site encoding amino acid residues 154-156. Hybrid PV156-204 and PV1-155 encoding cDNAs were prepared by ligation of the appropriate Eae I fragments of their parent cDNAs into pPICZαA.

[0202] (iv) PV1-8, PV1-18 and PV195-204. These hybrids were prepared by PCR with cDNA of Pol a 5 as the template. PV1-8 was prepared using primers 2 (SEQ ID NO: 32) and 6 (SEQ ID NO: 36). PV1-18 was prepared using primers 6 (SEQ ID NO: 36) and 13 (SEQ ID NO: 43). PV195-204 was prepared using primers 4 (SEQ ID NO: 34) and 14 (SEQ ID NO: 44). The hybrids were cloned into pPICZαA.

[0203] (v) PV1-24, PV1-32, PV1-39, PV1-50, PV1-57 and PV1-70. These hybrids were constructed using the PCR overlap extension method given in Example 1 (Ho et al., 1989, Gene 77:51). For PV1-24, first round PCRs were conducted using primers 1 (SEQ ID NO: 31) and 15 (SEQ ID NO: 45) with Ves v 5 cDNA as template and primers 6 (SEQ ID NO: 36) and 16 (SEQ ID NO: 46) with Pol a 5 cDNA as template. The two overlapping PCR products were then purified and used as template in a third PCR using flanking primers 1 (SEQ ID NO: 31) and 6 (SEQ ID NO: 36) to yield PV1-24. For PV1-32, first round PCRs were conducted using primers 1 (SEQ ID NO: 31) and 18 (SEQ ID NO: 48) with Ves v 5 cDNA as template and primers 6 (SEQ ID NO: 36) and 17 (SEQ ID NO: 47) with Pol a 5 cDNA as template. The two overlapping PCR products were then purified and used as template in a third PCR using flanking primers 1 (SEQ ID NO: 31) and 6 (SEQ ID NO: 36) to yield PV1-24. For PV1-39, first round PCRs were conducted using primers 2 (SEQ ID NO: 32) and 19 (SEQ ID NO: 49) with Ves v 5 cDNA as template and primers 6 (SEQ ID NO: 36) and 20 (SEQ ID NO: 50) with Pol a 5 cDNA as template. The two overlapping PCR products were then purified and used as template in a third PCR using flanking primers 2 (SEQ ID NO: 32) and 6 (SEQ ID NO: 36) to yield PV1-39. For PV1-50, first round PCRs were conducted using primers 2 (SEQ ID NO: 32) and 28 (SEQ ID NO: 58) with Ves v 5 cDNA as template and primers 6 (SEQ ID NO: 36) and 27 (SEQ ID NO: 57) with Pol a 5 cDNA as template. The two overlapping PCR products were then purified and used as template in a third PCR using flanking primers 2 (SEQ ID NO: 32) and 6 (SEQ ID NO: 36) to yield PV1-50. For PV1-57, first round PCRs were conducted using primers 2 (SEQ ID NO: 32) and 30 (SEQ ID NO: 60) with Ves v 5 cDNA as template and primers 6 (SEQ ID NO: 36) and 29 (SEQ ID NO: 59) with Pol a 5 cDNA as template. The two overlapping PCR products were then purified and used as template in a third PCR using flanking primers 2 (SEQ ID NO: 32) and 6 (SEQ ID NO: 36) to yield PV1-57. For PV1-76, first round PCRs were conducted using primers 2 (SEQ ID NO: 32) and 32 (SEQ ID NO: 62) with Ves v 5 cDNA as template and primers 6 (SEQ ID NO: 36) and 31 (SEQ ID NO: 61) with Pol a 5 cDNA as template. The two overlapping PCR products were then purified and used as template in a third PCR using flanking primers 2 (SEQ ID NO: 32) and 6 (SEQ ID NO: 36) to yield PV1-76. Hybrid cDNAs were cloned into pPICZαA.

[0204] (vi) PV22-32, PV15-125, PV142-150 and PV176-182. These constructs are hybrid Ag 5s wherein short Ves v 5 polypeptides replace homologous sequences in otherwise intact full length Pol a 5.

[0205] The Pol a 5 sequences were substituted with Ves v 5 sequences using the PCR overlap extension method given in Example 1 (Ho et al., 1989, Gene 77:51). The template DNA used for the first set of two PCRs was the Pol a cDNA of Lu et al. (1993, J. Immunol. 150:2823). The upstream and downstream Pol a primers used in the PCR extension protocols were primers 4 (SEQ ID NO: 22) and 6 (SEQ ID NO: 24), respectively. Final products were cloned into pPICZαA.

[0206] The overlapping primer pairs encoding the inserted Ves v 5 sequences were as follows: (a) PV22-32- primers 17 (SEQ ID NO: 47) and 18 (SEQ ID NO: 48) (b) PV115-125-primers 21 (SEQ ID NO: 51) and 22 (SEQ ID NO: 52)(c)PV142-150- primers 23 (SEQ ID NO: 53) and 24 (SEQ ID NO: 54) and (d) PV176-182- primers 25 (SEQ ID NO: 55) and 26 (SEQ ID NO: 56). PCR reaction and cycling conditions were those described for PV1-46.

4TABLE 1Primers for preparation of Ves v and Pol a 5s and their hybrids.PrimerSequence (5′ to 3′)1CGTGAATTCAACAATTATTGTAAAATAAAA(SEQ ID NO:31)2CGTCTCGAGAAAAGAAACAATTATTGTAAAATAAAA(SEQ ID NO:32)3CGTTCTAGATTACTTTGTTTGATAAAGTTC(SEQ ID NO:33)4CGTGAATTCGTTGATTATTGTAAAATAAAA(SEQ ID NO:34)5CGTCTCGAGAAAAGAGTTGATTATTGTAAAATAAAA(SEQ ID NO:35)6CGTTCTAGATTATTTTTTTGTATAAGGTAG(SEQ ID NO:36)7GTAAGCGAGCACAATCGGTTT(SEQ ID NO:37)8AAACCGATTGTGCTCGCTTAC(SEQ ID NO:38)9GTAGCAAAATTTCAGGTTGGA(SEQ ID NO:39)10TCCAACCTGAAATTTTGCTAC(SEQ ID NO:40)11ACCGCAAAATTTCCAGTTGGA(SEQ ID NO:41)12TCCAACTGGAAATTTTGCGGT(SEQ ID NO:42)13CGTGAATTCAACAATTATTGTAAAATAAAATGTTTGAAAGGAGGTGTCCATACTGCCT(SEQ ID NO:43)GCAAATATGGAGAA14CGTTCTAGATTACTTTGTTTGATAAAGTTCCTCATTCTTAAAATTTCCAGCTGG(SEQ ID NO:44)15GGCACAATTCTTGCTCGGTTTAAGACTTCCATA(SEQ ID NO:45)16TATGGAAGTCTTAAACCGAGCAAGAATTGTGCC(SEQ ID NO:46)17CTTAAACCGAATTGCGGTAATAAGGTAGTGGTATCGGTTGGTCCA(SEQ ID NO:47)18TGGACCAACCGATACCACTACCTTATTACCGCAATTCGGTTTAAG(SEQ ID NO:48)19TATGGTCTAACGAAACAAGAGAAAAAATTAATCGTA(SEC ID NO:49)20TACGATTAATTTTTTCTCTTGTTTCGTTAGACCATA(SEC ID NO:50)21TTAACAGGTAGCACGGCTGCTAAATACGATGATGTAGTCAGTCTA(SEQ ID NO:51)22ATCATCGTATTTAGCAGCCGTGCTACCTGTTAACGCTATATTTTG(SEQ ID NO:52)23CCTAAGAAAAAGTTTTCGGGAAACGACTTTGCTAAAATTGGC(SEQ ID NO:53)24GTCGTTTCCCGAAAACTTTTTCTTAGGATTAAAATCTTTCAC(SEQ ID NO:54)25ATTCAAGAGAAATGGCACAAACATTACCTCATA(SEQ ID NO:55)26TTTGTGCCATTTCTCTTGAATATATTTTAGAGA(SEQ ID NO:56)27GAGCACAATGACTTTAGACAAAAA(SEQ ID NO:57)28TTTTTGTCTAAAGTCATTGTGCTC(SEQ ID NO:58)29AAAATTGCACGAGGGTTGGAAACA(SEQ ID NO:59)30TGTTTCCAACCCTCGTGCAATTTT(SEQ ID NO:60)31AATATGAAAAATTTGGTATGGAAC(SEQ ID NO:61)32GTTCCATACCAAATTTTTCATATT(SEQ ID NO:62)

[0207] Ag5- or hybrid-encoding cDNAs of the EA- or KR-series were digested, respectively, with restriction enzymes Eco RI or Xho I, and Xba I, then inserted into similarly cut pPICZα-A vector (Invitrogen, San Diego, Calif.). The recombinant plasmids were amplified in TOP10F′ cells. The Ag 5-coding sequences of all recombinant plasmids were confirmed by DNA sequencing. The Ag 5 coding-sequences corresponded to the sequence data in Genbank (Accession number M98858 for Ves v Ag 5 and accession number M98857 for Pol a Ag 5), with the exceptions of two single-nucleotide differences observed for Ves v 5. These changes were at positions 579 and 587 and resulted, respectively, in a silent G to A mutation and a T to A substitution that resulted in a codon change of M to K at amino acid residue 196. The two nucleotide changes may represent insect polymorphism, rather than random mutations since the Ag 5 cDNAs used were prepared in the same manner as it was done previously (Lu et al., 1993, J. Immunol. 150:2823).

EXAMPLE 2

Expression and Purification of Ag 5s and Hybrids

[0208] Recombinant plasmids (1-2 μg) were linearized by cutting with the restriction enzyme Sac I then used to transform competent Pichia pastoris KM71 yeast cells (about 8×109 cells in 40 μl of 1 M sorbitol) by electroporation. Transformed cells were diluted to 2 ml with 1 M sorbitol and allowed to recover at 30° for 1 hr without shaking and for an additional hour with shaking at 200 rpm. Aliquots of 50 μl or 100 μl aliquots were then spread on 100 mm plates of YPDS medium containing 1.5 mg/ml Zeocin for selection of multi-copy integrants (Invitrogen Manual). Selected clones were picked after 3-4 day incubation and screened by small scale expression to identify colonies producing hybrid protein. Small scale expression was carried out in 50 ml plastic tubes in the same manner as described below for large scale isolation but at {fraction (1/30)} scale and the culture fluids were screened by SDS gel electrophoresis for secreted proteins.

[0209] Yeast cells from selected clones were grown in two 500 ml bottles, each containing 150 ml of pH 6.0 phosphate buffer containing yeast nitrogen base, biotin, glycerol and histidine at 30° with orbital shaking at 250 rpm to an A600 nm of 10-12. Cells were then collected by centrifugation and resuspended in 100 ml of similarly buffered medium containing methanol in place of glycerol. Incubation was continued at 30° with shaking at 250 rpm for 4-6 days with daily addition of 1 ml of 50% methanol.

[0210] Ag 5s or their hybrids were purified from the culture fluid concentrate by ion-exchange chromatography on SE-cellulose (Sigma) using a previously reported procedure (Monsalve et al., 1999, Protein Expr. Purif. 16:410). About 70% of the main peak was pooled, desalted by reversed phase chromatography on C18 silica and lyophilized. Recombinant Ag 5s or hybrids were dissolved in 0.01 M ammonium acetate buffer (pH 4.6) and stored at 4°. Recombinant protein concentrations were determined from absorbance at 280 nm, using molar extinctions calculated from tyrosine and tryptophan contents. The yields of Ag 5s or hybrids typically ranged from 1 to 7 mg per 100 ml of 4-day cultures.

[0211] Recombinant Ag 5s or hybrids were characterized by SDS gel electrophoresis, N-terminal sequence analysis and MALDI mass spectrometry. CD spectra at 0.2 mg/ml of recombinant proteins in 0.01 M acetate buffer of pH 4.6 were taken in cells of 1 mm path length in an AVIV 62DS spectrometer.

EXAMPLE 3

Physico-Chemical Characterization of Recombinant Vespid Ag 5s and Hybrids

[0212] The Ag5s and hybrid proteins expressed in yeast strain KM71 contained a secretory signal peptide. The signal peptide was linked to the expressed protein via a peptide of KR or KREAEAEF sequence. These two types of proteins were designated as the KR- and EA-series, respectively. Upon secretion from the yeast cells, the signal peptide was cleaved from the secreted protein at the KR sequence (Kex 2 protease site) or the two EA sequences (Ste 13 dipeptidyl amino peptidase sites) (Invitrogen Manual).

[0213] Recombinant proteins were isolated from culture fluid by ion exchange chromatography on SE-cellulose followed by reversed phase chromatography on C18-silica and characterized by SDS gel electrophoresis. (FIG. 6). Several hybrids showed a closely-spaced doublet with mobilities similar to that of natural Ves v 5. The doublets are consistent with the varying extents of processing at their N-terminal ends, as indicated by N-terminal sequencing of hybrids PV1-155 and PV156-204 and mass spectrometry data (Table 2).

[0214] Recombinant Ag 5s and hybrids showed nearly identical CD spectra as those of the natural Ag 5s (FIG. 7). The spectra of the natural Ves v 5 and the EA-Ves v 5, and those of EA-PV1-46, EA-PV1-155 and EA-PV156-204 showed the presence of minima at about 208 nm with a shoulder at 225 nm (FIG. 7). These features are indicative of an ordered feature (Yang et al., 1986, Methods in Enzymology 130:208). Similar CD spectra were observed for the other hybrids listed in Table II (data are not shown). The CD spectrum of recombinant Ves v 5 from bacteria showed a minima at about 200 nm, which is indicative of a disordered structure (Monsalve et al., 1999, Protein Expr. Purif. 16:410).

[0215] The recombinant Ag 5s and hybrids from yeast were freely soluble in acid or basic buffers, as were the natural Ag 5s. This is in contrast to recombinant vespid Ag 5s from bacteria, which were freely soluble only in acidic buffer.

[0216] Results of mass spectrometric analysis of Ag 5s and hybrids are given in Table 2. EA-series Ag 5s were cleaved efficiently at the Kex 2 site but showed variable cleavages at the two Ste 13 sites. Recombinant EA-series proteins, therefore, had amino-terminal sequences of EAEAEF and EAEF, where the EF sequence was encoded by the Eco R I site used to insert cDNA into the vector. These data were similar to results reported previously (Monsalve et al., 1999, Protein Expr. Purif. 16:410).

[0217] The EAEAEF sequence of recombinant Ves v 5 is known to function as a strong hapten (Monsalve et al., 1999, Protein Expr. Purif. 16:410). Therefore, Ag 5s were also expressed as KR-series hybrids. Cleavage of KR-series proteins at the Kex 2 site yielded recombinant proteins with the N-terminal sequence of the natural proteins. Mass spectrometry analysis of the KR-series proteins Ves v 5, Pol a 5, and hybrids KR-PV1-24 and KR-PV1-46 showed that they were cleaved, with varied efficiencies, at the Kex2 site, and at residues 2, 7, and 9 upstream of the Kex2 site. (Table 2.) The recombinant proteins of the KR-series were usually of slightly lower yields than those of the EA-series.

5TABLE 2Mass spectrometric data of recombinant vespid Ag 5s and hybrids.Mass unitsProteinAssumed sequenceAbundance1calc'dfoundEA-Ves v 5EAEAEF-Vv80%23,95423,947EAEF-Vv20%23,75423,752EA-Pol a 5EAEAEF-Pa100%23,61123,613EA-PV1-18EAEF-PV43%23,49723,506EAEAEF-PV36%23,69723,698REAEAEF-PV21%23,87123,827EA-PV1-18EAEAEF-PV100%23,69723,701EA-PV1-32EF-PV60%22,96422,930EAEF-PV40%23,15123,134EA-PV1-46EAEF-PV53%23,30023,327EAEAEF-PV47%23,50023,515EA-PV1-46EF-PV10%23,09923,109EAEF-PV50%23,30023,327EAEAEF-PV40%23,50023,515EA-PV1-155EF-PV53%23,37523,334EAEF-PV47%23,57523,533EA-PV22-32EAEF-PV55%23,13523,203EAEAEF-PV45%23,33623,371EA-PV115-125EAEAEF-PV100%23,87323,887EA-PV142-150EAEAEF-PV100%23,59223,585EA-PV156-204EAEF-PV59%23,77623,775EAEAEF-PV41%23,93223,939EA-PV195-204EAEAEF-PV70%23,70023,688REAEAEF-PV30%23,87423,844KR-Ves v 5Vv590%23,27723,274EEGVSLEKR-Vv10%24,30524,298KR-Ves v 5Vv95%23,27723,284EEGVSLEKR-Vv5%24,30524,300KR-Pol a 5Pa20%22,93422,951EEGVSLEKR-Pa80%23,96223,992KR-Pol a 5Pa10%22,93422,935EEGVSLEKR-Pa90%23,96223,962KR-PV1-24PV85%22,90322,897EEGVSLEKR-PV15%23,93123,933KR-PV1-46PV70%22,82322,834KR-PV30%23,10723,157KR-PVL-46PV60%22,82322,834KR-PV40%23,10723,1571Protein abundance was estimated from peak heights of samples in mass spectra. N-terminal sequences For of EA-Ves v 5, EA-Pol a 5, EA-PV3PV156-204 and EA- VP3PV1-155 samples of EA series, their assumed sequences were confirmed by Edman degradation. Results of two preparations are shown for each of EA-PV1-18, EA-PV1-46, KR-Ves v 5, KR-Pol a and KR-PV1-46.

[0218] Amino terminal peptides have been assigned SEQ ID NO: as follows; EAEAEF [SEQ ID NO: 89]; EAEF [SEQ ID NO: 90]; REAEAEF [SEQ ID NO: 91] and EEGVSLEKR [SEQ ID NO: 92].

EXAMPLE 4

ELISA Studies

[0219] ELISA was performed in 96-well plates in the wells coated with 4 μg/ml Ag 5 in 0.05 M Tris-HCI buffer of pH 8. Bound IgG1 was detected with 2 μg/ml biotinylated goat anti-mouse IgG (γ1 specific) followed with 2 μg/ml avidin-peroxidase conjugate (King et al., 1995, J. Immunol 154:577). Antibody concentrations of sera samples were determined by comparison of their ELISA data with that of an immuno-affinity purified sample of Ves v 5-specific antibody.

EXAMPLE 5

Ves v 5-Specific B Cell Epitopes of Hybrids

[0220] Murine polyclonal antibodies specific for natural Ves v 5 were isolated from BALB/c sera by affinity chromatography on Ves v 5-specific immunosorbent and were depleted of Pol a 5-cross-reacting antibodies by passage through Pol a 5-specific immunosorbent. The immunosorbents were prepared with CNBr activated Sepharose 2B (Pharmacia). Murine monoclonal antibodies specific for Ves v 5 were obtained as described (King et al., 1987, Mol. Immunol 24:857).

[0221] Ves v 5-specific B cell epitopes were detected by hybrid-inhibition of binding of mouse Ves v 5-specific antibodies to solid-phase Ves v 5. Both EA- and KR-Ves v 5 were tested as solid phase antigen with similar results. Five samples of mouse antisera were tested; three were from BALB/c strains and one each from ASW/sn and P/J strains. Results using one BALB/c serum sample are shown in FIG. 8A. At the highest concentration of 50 or 500 μg/ml inhibitor tested, the two N-terminal hybrids EA-PV1-46 and EA-1-155 showed maximal inhibition approaching 100%, as did EA- or KR-Ves v 5. Two other N-terminal hybrids KR-PV1-24 and EA-PV1-32 had maximal inhibition of about 60% and the shortest N-terminal hybrid, EA-PV1-18, had maximal inhibition of about 20%. The C-terminal hybrid EA-PV156-204 had maximal inhibition of about 15%. Similar results were obtained for results of inhibition ELISA using antisera from ASW/sn (FIG. 8B) and P/J (FIG. 8C) mice.

[0222] Ves v 5-specific B cell epitopes were also detected by inhibition analyses with sera from six yellow jacket sensitive patients. The data from three patients are shown in FIG. 9A-C. The results were similar to those obtained with mouse IgGs.

[0223] The results of the ELISA inhibition studies using both mouse and human antisera indicated the immunodominance of the N-terminal region of Ves v 5.

[0224] The observed inhibition by the hybrids was not due to cross-reacting epitopes of the Pol a 5 portion of the molecule as the sample of Ves v 5-specific antibodies used for inhibition studies in BALB/c mice was depleted of Pol a 5-cross-reactive antibodies and no inhibition by Pol a 5 was detected (FIG. 8A). The high concentrations of hybrids required for half maximal inhibition relative to that of Ves v 5 did not reflect that the epitopes of the hybrids lacked the native structure of Ves v 5 as the recombinant Ves v 5 from bacteria that lacked the native structure did not show any inhibition (data not shown).

[0225] The difference in the inhibitory activities of Ves v 5 and hybrids was probably related to their epitope densities. Epitope density is known to influence strongly the affinity constant of a multivalent antigen and a bivalent antibody (Hornick and Karush, 1972, Immunochemistry 9:325; Crothers and Metzger, 1972, Immunochemistry 9:341).

[0226] The data in FIGS. 8 and 9 suggested that the amino terminal portion of Ves v 5 includes the immunodominant B cell epitopes of Ves v 5. This finding was confirmed by tests with a panel of 17 monoclonal antibodies specific for Ves v (King et al., 1987, Mol. Immunol 24:857). These monoclonal antibodies were specific for the natural Ves v 5 and recombinant proteins from yeast, but they did not bind the denatured form of recombinant Ves v 5 from bacteria (data not shown). ELISA results showed that one monoclonal antibody bound EA-Ves v 5 and EA-PV1-46 with similar affinity and maximal binding and it did not bind any of the other N- or C-terminal hybrids (FIG. 10A). Four other monoclonal antibodies showed greatly reduced maximal binding to EA-PV1-46 but no binding to any of the shorter N-terminal hybrids; the data for one such antibody are given in FIG. 10B. Lastly, one monoclonal antibody showed greatly reduced binding to EA-PV1-32 and EA-PV 1-46 and moderate binding to EA-PV1-18 and EA-PV 1-24 (FIG. 10C). These data show that six of the 17 monoclonal antibodies tested were specific for the N-terminal region of Ves v 5.

EXAMPLE 6

Immune Responses to Hybrids

[0227] Groups of 3 or 4 female BALB/c mice were given biweekly intraperitoneal injections of 2 μg immunogen and 1 μg alum in 0.2 ml of phosphate buffered saline. Ag 5 or hybrid specific sera were collected at week 5 or later. Similar antibody levels were observed for sera collected at weeks 5, 7, and 9.

[0228] Mice immunized with hybrids produced antibodies specific for the hybrid, Pol a 5 and Ves v 5. The antibody levels of sera samples were measured before and after absorption with Pol a 5 to determine their specificity for Ves v 5. These data are summarized in Table 3A . Mice immunized with natural, EA- or KR-Ves v 5 gave nearly the same antibody responses, and only those of the KR-Ves v 5 are given Table 3A. EA-PV1-46 gave a higher antibody response in set A mice than KR-PV1-46 did in set B mice. This difference may be due to the different sets of mice used. EA-PV 1-18 was used in both sets of experiments, and it gave higher antibody response in set A mice than that in set B mice.

[0229] Comparison of antibody levels in the N-terminal hybrid-specific sera samples in Table 3, before and after Pol a 5 absorption, indicated that 30-80% of the antibodies were specific for Ves v 5 when tested on solid-phase Ves v 5, and these values were less when tested on solid-phase hybrid. The higher contents of Ves v 5-specific antibodies detected on solid-phase Ves v 5 than those on solid-phase hybrid suggest that the majority of hybrid-specific antibodies recognize overlapping regions of Ves v 5 and Pol a 5 in the hybrid. The data in set A of Table 3A indicated that of the three N-terminal hybrids, PV1-155 was as immunogenic as Ves v 5 was, PV1-46 was half as immunogenic as Ves v 5 and PV1-18 was about {fraction (1/9)}th as immunogenic as Ves v 5. The data in set B indicate that PV1-46 and 1-32 were more immunogenic than PV1-24 and 1-18. The data from both sets suggest that the longer N-terminal hybrids PV1-46 and 1-32 stimulate higher contents of Ves v 5-specific antibodies and lower contents of Pol a 5- specific antibodies than the two shorter hybrids PV1-24 and 1-18 did.

6TABLE 3AMurine antibody responses to vespid antigen 5s and hybridsmg/ml specific IgG in seraby ELISA on solid-phase2,3SETImmunogen1EA-Ves v 5EA-Pol a 5HybridAKR-Ves v 5 8.9 (8.5)0.6—KR-Pol a 5 2.8 (1.0)7.0—EA-PV1-15512.00.7—EA-PV1-46 4.2 (3.5)1.9 7.6 (5.6)EA-PV1-18 1.0 (0.8)6.9 6.9 (0.7)EA-PV156-204 1.6 (0.6)10.0 2.6 (0.3)EA-PV195-204 1.3 (0.4)14.010.0 (0.3)BKR-Ves v 515.0(14.0)0.2—KR-PV1-46 0.6 (0.5)1.0 2.7 (3.0)EA-PV1-32 0.9 (0.7)4.3 8.0 (3.2)KR-PV1-24 0.4 (0.3)4.2 6.5 (0.9)EA-PV1-18 0.4 (0.3)4.5 5.3 (0.7)

[0230] 1. Sera were collected on week 7, after 3 biweekly ip injections of immunogen. Sets A and B studies were made at separate occasions.

[0231] 2. Antibody concentration was estimated from reciprocal sera concentration required to give an absorbance change of 1.0 in 30 minutes. Under the conditions used, this change corresponded to a 0.1 μg/ml solution of purified Ves v 5- specific antibody. The estimated antibody concentrations varied by about 40% on repeat measurements.

[0232] 3.Values in parenthesis were obtained after absorption of {fraction (1/500)} diluted sera with 0.2 mg/ml EA-Pol a 5.

[0233] The results shown in Table 3A indicate the B cell epitope of Ves v 5 is in its N-terminal region. Additional hybrids of Ves v 5 and Pol a 5 were prepared and tested for immunogenicity in mice as described above, to delineate the N and the C-terminal limits of the dominant B cell epitope region. Results are given in Table 3B, which lists the IgG1 content specific for Ves v, Pol a or hybrid, and percent of specific IgG1 remaining after absorption with Pa.

[0234] Hybrid PV1-8 with the lowest Ves v content did not induce Ves v-specific antibody response. All other hybrids induced 0.4-4.5 mg/ml of Ves v-specific Ab with the exception of PV22-32. Hybrids with Ves v contents <PV1-32 are moderately specific for Ves v response, as 34-81% of their Ves v-specific antbody and 15-27% of their hybrid-specific antibodies were not absorbed by Pol a 5. Hybrids with Ves v contents >PV1-39 are more specific, as 66-96% of their Ves v 5-specific antibody and 91-100% of their hybrid-specific antibody were not absorbed by Pol a 5. These results together suggest the C-terminal limit of the dominant epitope region is between residues 32-39.

[0235] Hybrids with Ves v contents of <PV1-32 show 2-4 mg/ml of Pol a-specific antibody, and hybrids with Ves v contents of >PV1-39 showed 0.04-1.34 mg/ml of Pol a-specific antibody. As the Ves v content of hybrids was increased from PV1-32 to 1-76, there was a progressive decrease of Pol a-specific response. These results together suggest the C-terminal limit of the dominant epitope extends beyond residues 39, as suggested by considerations of the Ves v-specific response to hybrids.

[0236] The lack of Ves v-specific antibody response of PV1-8 and 22-32 as compared to the response of PV1-32 suggests the N-terminal limit of the dominant epitope region to be within residues 9-21.

7TABLE 3BMurine antibody responses to vespid antigen 5s and hybridsGroupsVes v 5 specificHybrid specific IgG1;Constructof miceIgG1; % Ves vPol a 5 specific IgG1% Ves vPol a 511.80 mg/ml; 64%4.50 mg/mlVes v 5410.7 ± 3.2 mg/ml0.2 ± 0.1 mg/ml104 ± 15%PV1-8108.2 mg/mlPV1-1840.6 ± 0.44 mg/ml;4.1 ± 2.0 mg/ml7.5 ± 4.5 mg/ml;68 ± 14%27 ± 26%PV1-2420.35 ± 0.06 mg/ml;2.26 ± 0.50 mg/ml5.86 ± 1.30 mg/ml;81 ± 17%20 ± 12%PV1-3230.52 ± 0.39 mg/ml;3.77 ± 1.89 mg/ml6.82 ± 3.46 mg/ml;34 ± 25%15 ± 6%PV1-3924.45 ± 0.70 mg/ml;1.72 ± 0.06 mg/ml8.25 ± {fraction (1/87)} mg/ml;89 ± 27%76 ± 23%PV1-4632.29 ± 3.41 mg/ml;1.18 ± 0.96 mg/ml7.57 ± 7.33 mg/ml;86 ± 14%91 ± 9%PV1-5011.01 mg/ml; 94%0.44 mg/ml11.22 mg/ml; 90%PV1-5710.67 mg/ml; 96%0.22 mg/ml11.88 mg/ml; 85%PV1-7611.32 mg/ml; 92%0.04 mg/ml11.88 mg/ml; 92%PV22-3210.04 mg/ml; 0%4.88 mg/ml 6.31 mg/ml; 6%

[0237] Data are from averages of week 7 bleedings from 1-4 groups of 4 mice. % Ves v refers to antibody content after absorption with Pol a 5

EXAMPLE 7

T Cell Response

[0238] Proliferation assays were performed with spleen cells from mice immunized with vespid antigen 5 or hybrid to study the specificity of T cell responses. Assays were performed in triplicate with spleen cells pooled from 2 to 3 mice, 10 days after 5 biweekly immunizations. Spleen cells (4×105) were cultured with test antigen in 0.2 ml of culture medium at 37° and 5% CO2. Tritiated thymidine (1 μCi) was added on day 3, and the thymidine uptake was determined on day 4. The results were expressed as stimulation index values.

[0239] Results showed that the hybrids EA-PV1-46, EA-PV1-155 and EA-PV156-204 induced hybrid-specific as well as vespid antigen 5-specific T cell responses (Table 4). The data indicated that the best proliferative responses were obtained when the stimulating antigen was the immunogen. This is apparent from comparing the maximal stimulation index values at the highest antigen concentration of 100 μg/ml tested, and from comparing the lowest antigen concentration required for a stimulation index value of 4.

8TABLE 4Vespid antigen 5 or hybrid stimulated proliferation of murine spleen cellsSpleen cellsStimulating Agspecific forEA-Ves v5EA-Pol a 5EA-hybridStimulation Index at 100 μg/ml AgKR-Ves v 58.21.5—KR-Pol a 52.26.3—EA-PV1-1556.12.25.0EA-PV1-466.08.013.5EA-PV1-182.35.06.1EA-PV156-2044.14.26.8EA-PV195-2041.78.64.1μg/ml Ag for stimulation index of 4KR-Ves v 52.6>100—KR-Pol a 5>10016—EA-PV1-15511>1000.54EA-PV1-46202.20.26EA-PV1-18>1004719EA-PV156-20460702.3EA-PV195-204>100882Background proliferation of spleen cells showed 3H-thymidine uptake of 400-900 cpm.

EXAMPLE 8

Allergenicity of Recombinant Vespid Ag 5s and Hybrids in Patients

[0240] Allergenicity was determined by histamine release assay from basophils of 10 yellow jacket sensitive patients, following challenge with Ag 5 or hybrids (Colombo et al., 1995, J Allergy Clin. Imm. 95:565). The patients/results shown in Table 5 are divided into two groups. Group A patients (n=7) were about 1000 times more sensitive to Ves v 5 than to Pol a 5; Group B patients (n=3) were about equally sensitive to both antigen 5s.

9TABLE 5Summary of histamine release data of hybridsReciprocal Activity Relative to Ves v 5Group AGroup BNo. ofNo. ofAllergenpatientsMeanRangepatientsMeanRangeVes v 5711311Pol a 571154 330-550030.70.2-2 PV1-155311-2211PV1-465126 13-330020.70.1-5 PV1-183583 12-5000224 3.0-200 PV22-32332072000-500026 6-20PV115-125332072000-500025 2-15PV142-150330002700-500025 2-15PV156-204611391000-3000330.4-70 PV195-20433207 50-500023220.0-50

[0241] The complete data from one patient of each group are given in FIG. 11.

[0242] Of the three N-terminal hybrids tested, EA-PV1-155 showed no decrease in allergnenicity. EA-PV1-46 and 1-18 showed geometric mean reductions of 126- and 583-fold respectively in group A patients, and 0.7- and 24-fold decreases respectively in group B patients. The two C-terminal hybrids EA-PV156-204 and 195-204 had reductions of 1139- and 3207-fold in group A patients respectively and 3- and 32-fold in group B patients respectively.

[0243] The different extents of reduction in allergenicity of the N- and C-terminal hybrids reflect both their IgE antibody concentration and their epitope density. The inhibition ELISA data in FIG. 6 suggest a higher concentration of human IgG antibodies for the N-terminal region of Ves v 5 than those for the C-terminal region and this is likely also the case for IgE antibodies. Another contributing factor to the greater reduction in allergenicity of the C-terminal hybrid EA-PV156-204 as compared to the N-terminal hybrid EA-PV1-46 is probably due to its decreased epitope density as the C-terminal hybrid has fewer surface accessible residues of Ves v 5 than the N-terminal hybrid does. Similarly, the greater reduction in allergenicity of the shorter N- or C-terminal hybrids, PV1-18 or PV195-204, as compared to their respective longer ones also reflects the influence of epitope density.

[0244] The allergenicity of recombinant Ves v 5 from bacteria was compared with those of the natural Ves v and the recombinant Ves v 5 from yeast. In three patients tested, the recombinant protein from bacteria was about 103 times less potent than the natural protein or the recombinant protein from yeast (data not shown). These data confirm previous observations that the majority of B cell epitopes for allergens are dependent on the conformation of the native allergen (King et al., 2000, Int Arch Allergy 123:99).

[0245] The decrease in allergenicity of the recombinant Ves v 5 from bacteria, was due to loss of the conformation dependent B cell epitopes as the CD spectrum of the recombinant protein from bacteria showed it to have a disordered structure. However, the decrease in allergenicity of the hybrid protein PV1-46 or PV156-204 was due to reduction of the number and density of Ves v 5-specific epitopes, as its CD spectrum indicated it had an ordered structure similar to that of Ves v 5. The reduction of the number and density epitopes of the hybrid PV1-46 and PV156-204 is in agreement with the B cell epitope and immunogenicity data given in Examples 5-7.

EXAMPLE 9

Crystallization of Recombinant Ves v 5

[0246] Crystals of Ves v 5 was grown by the vapor diffusion technique at 25° C. For crystallization, 5 μl of 5 mg/ml Ves v 5 was mixed with 5 μl of 18% PEG 6000, 0.1 M sodium citrate, pH 6.0 and equilibrated against 1 ml of 18% PEG 6000, 0.1 M sodium citrate, pH 6.0. X-ray diffraction data was collected at 100K from native Ves v 5 crystals and after incorporation of heavy-atom derivatives and used to solve the three-dimensional structure of Ves v 5. The atomic coordinates and structure factors of Ves v 5 have been deposited in the Protein Data Bank (PDB) with the accession number Q05110. The atomic coordinates of Ves v 5 are given in Table 6.

10TABLE 6Yes v 5 crystal coordinatesREMARK FILENAME = “brefinement.pdb”REMARK r = 0.215955 free_r = 0.29 REMARK DATE: 28 Oct. 199815:45:46created by user: anetteATOM1CBGLU117.07751.79323.6621.0041.80APEPATOM2CGGLU116.59552.04725.0811.0043.97APEPATOM3CDGLU115.16751.58025.3101.0044.74APEPATOM4OE1GLU114.36751.64024.3521.0046.38APEPATOM5OE2GLU114.84551.15626.4441.0043.48APEPATOM6CGLU119.16950.42923.6641.0039.72APEPATOM7OGLU119.73349.57524.3581.0040.19APEPATOM8NGLU117.00549.43124.4041.0041.50APEPATOM9CAGLU117.65550.39123.4581.0040.85APEPATOM10NALA219.82051.42323.0641.0037.33APEPATOM11CAALA221.26751.57123.1791.0034.17APEPATOM12CBALA221.66851.73524.6571.0034.25APEPATOM13CALA221.93550.34122.5851.0032.32APEPATOM14OALA221.29949.58021.8471.0033.01APEPATOM15NGLU323.21550.14822.8991.0029.81APEPATOM16CAGLU323.95648.99122.4021.0026.33APEPATOM17CBGLU324.94849.41321.3251.0030.89APEPATOM18CGGLU325.24648.32020.3031.0035.96APEPATOM19CDGLU324.02947.46819.9731.0038.25APEPATOM20OE1GLU323.42847.67818.8911.0039.27APEPATOM21OE2GLU323.68146.58620.7931.0037.45APEPATOM22CGLU324.69348.26923.5301.0021.89APEPATOM23OGLU325.78048.67923.9591.0020.16APEPATOM24NALA424.09347.18023.9951.0017.32APEPATOM25CAALA424.65246.38225.0801.0015.71APEPATOM26CBALA423.79645.14125.3021.0012.64APEPATOM27CALA426.10345.97024.8621.0014.17APEPATOM28OALA426.81645.71025.8271.0011.99APEPATOM29NGLU526.54245.90823.6031.0012.66APEPATOM30CAGLU527.91745.50323.3191.0013.51APEPATOM31CBGLU528.22245.58321.8171.0015.08APEPATOM32CGGLU529.64745.12721.4791.0020.49APEPATOM33CDGLU530.06845.44720.0491.0022.60APEPATOM34OE1GLU529.22445.94819.2781.0024.69APEPATOM35OE2GLU531.24545.19919.6991.0023.87APEPATOM36CGLU528.94946.33924.0651.0012.46APEPATOM37OGLU530.02545.84724.3941.0012.28APEPATOM38NPHE628.61647.59624.3431.0011.87APEPATOM39CAPHE629.54648.49125.0221.0011.93APEPATOM40CBPHE629.45949.87924.3771.0012.32APEPATOM41CGPHE629.70649.85722.8871.0014.45APEPATOM42CD1PHE628.64649.80321.9971.0014.86APEPATOM43CD2PHE631.00149.81122.3811.0014.25APEPATOM44CE1PHE628.87049.69820.6231.0015.78APEPATOM45CE2PHE631.23649.70521.0081.0013.92APEPATOM46CZPHE630.16649.64820.1311.0013.36APEPATOM47CPHE629.37848.55626.5371.0010.13APEPATOM48OPHE629.89249.46327.2011.009.26APEPATOM49NASN728.65847.56827.0661.0010.89APEPATOM50CAASN728.41147.42228.4981.007.63APEPATOM51CBASN727.04046.78628.7501.006.94APEPATOM52CGASN725.89747.77428.6581.005.91APEPATOM53OD1ASN726.04948.95328.9621.006.68APEPATOM54ND2ASN724.73547.28628.2401.002.00APEPATOM55CASN729.47746.42828.9291.008.03APEPATOM56OASN729.71245.44828.2231.007.49APEPATOM57NASN830.12646.66330.0661.007.97APEPATOM58CAASN831.15545.73530.5361.009.65APEPATOM59CBASN832.19346.46931.3841.0011.85APEPATOM60CGASN833.24145.53131.9611.0013.69APEPATOM61OD1ASN833.49344.45931.4151.0012.11APEPATOM62ND2ASN833.85845.93533.0711.0012.79APEPATOM63CASN830.55344.58631.3501.0010.91APEPATOM64OASN830.39744.69032.5641.0011.39APEPATOM65NTYR930.22543.49030.6741.0010.20APEPATOM66CATYR929.63142.33131.3281.009.11APEPATOM67CBTYR928.95641.43130.2871.008.55APEPATOM68CGTYR927.72742.05429.6891.006.89APEPATOM69CD1TYR927.79842.80528.5171.008.12APEPATOM70CE1TYR926.66843.42327.9911.009.63APEPATOM71CD2TYR926.49841.93230.3181.007.93APEPATOM72CE2TYR925.36242.54329.8061.009.55APEPATOM73CZTYR925.45243.28628.6461.0010.64APEPATOM74OHTYR924.32543.89328.1491.0011.41APEPATOM75CTYR930.62841.50932.1311.0010.32APEPATOM76OTYR930.23740.58432.8401.008.46APEPATOM77NCYS1031.91241.83432.0171.0011.72APEPATOM78CACYS1032.93441.09832.7501.0013.13APEPATOM79CCYS1032.83241.40434.2401.0014.57APEPATOM80OCYS1033.56540.83535.0511.0014.20APEPATOM81CBCYS1034.32941.47132.2421.0014.59APEPATOM82SGCYS1034.74740.86230.5691.0013.90APEPATOM83NLYS1131.91342.30034.5931.0015.58APEPATOM84CALYS1131.70642.69535.9821.0017.16APEPATOM85CBLYS1131.51444.21336.0731.0017.37APEPATOM86CGLYS1132.80545.02035.9081.0019.88APEPATOM87CDLYS1133.87944.54936.8721.0019.32APEPATOM88CELYS1135.25244.99436.4421.0022.07APEPATOM89NZLYS1136.14843.82436.2121.0026.09APEPATOM90CLYS1130.50341.98736.6001.0018.39APEPATOM91OLYS1130.33041.99037.8221.0018.93APEPATOM92NILE1229.67641.38235.7481.0017.37APEPATOM93CAILE1228.48840.66236.1971.0017.54APEPATOM94CBILE1227.52240.34835.0111.0015.92APEPATOM95CG2ILE1226.34739.50735.4971.0014.62APEPATOM96CG1ILE1227.03341.64534.3531.0014.71APEPATOM97CD1ILE1226.19742.54335.2461.0014.44APEPATOM98CILE1228.90239.33136.8171.0018.50APEPATOM99OILE1229.88438.72836.4011.0019.73APEPATOM100NLYS1328.14438.88437.8131.0019.79APEPATOM101CALYS1328.39137.60538.4681.0021.47APEPATOM102CBLYS1328.97837.81139.8711.0024.55APEPATOM103CGLYS1328.34938.95940.6641.0029.46APEPATOM104CDLYS1329.13939.27241.9341.0032.01APEPATOM105CELYS1329.96640.54641.7861.0034.07APEPATOM106NZLYS1330.86740.51640.5911.0034.69APEPATOM107CLYS1327.05136.86738.5551.0020.70APEPATOM108OLYS1326.05037.43338.9761.0019.96APEPATOM109NCYS1427.02935.61138.1321.0020.06APEPATOM110CACYS1425.80834.83138.1761.0020.78APEPATOM111CCYS1425.74134.06239.4821.0022.64APEPATOM112OCYS1426.72433.99440.2181.0022.31APEPATOM113CBCYS1425.75233.87536.9871.0019.10APEPATOM114SGCYS1425.35234.72435.4221.0016.84APEPATOM115NLEU1524.57733.49239.7751.0024.99APEPATOM116CALEU1524.40032.74641.0151.0027.03APEPATOM117CBLEU1522.95332.25141.1381.0027.78APEPATOM118CGLEU1522.05432.96342.1521.0028.08APEPATOM119CD1LEU1520.69932.26942.1941.0028.30APEPATOM120CD2LEU1522.69932.95343.5351.0027.17APEPATOM121CLEU1525.36531.57441.0901.0027.24APEPATOM122OLEU1526.06531.40242.0881.0028.76APEPATOM123NLYS1625.41030.77440.0331.0028.73APEPATOM124CALYS1626.30029.62140.0051.0030.04APEPATOM125CBLYS1625.67928.47839.2011.0031.71APEPATOM126CGLYS1624.16228.40139.2711.0032.24APEPATOM127CDLYS1623.56227.75738.0091.0033.96APEPATOM128CELYS1624.53627.73836.8201.0033.82APEPATOM129NZLYS1623.82827.60435.5151.0033.08APEPATOM130CLYS1627.65929.96639.4171.0030.04APEPATOM131OLYS1628.44229.07139.0921.0031.31APEPATOM132NGLY1727.93331.26139.2731.0029.07APEPATOM133CAGLY1729.21431.69838.7441.0027.07APEPATOM134CGLY1729.41031.55337.2431.0026.38APEPATOM135OGLY1728.44831.55236.4721.0025.25APEPATOM136NGLY1830.67031.42836.8311.0025.19APEPATOM137CAGLY1830.98331.29435.4201.0022.24APEPATOM138CGLY1831.13932.65534.7711.0020.24APEPATOM139OGLY1830.51033.62235.1951.0021.83APEPATOM140NVAL1931.97432.73533.7431.0016.65APEPATOM141CAVAL1932.21233.98933.0401.0015.58APEPATOM142CBVAL1933.51633.89632.2221.0015.68APEPATOM143CG1VAL1933.88435.25431.6491.0013.84APEPATOM144CG2VAL1934.63333.36433.1081.0015.09APEPATOM145CVAL1931.04534.36132.1151.0014.11APEPATOM146OVAL1930.62233.56231.2781.0014.03APEPATOM147NHIS2030.52835.57732.2651.0011.37APEPATOM148CAHIS2029.41036.02031.4441.0011.65APEPATOM149CBHIS2029.09437.49331.7041.0012.93APEPATOM150CGHIS2027.72137.90031.2641.0013.85APEPATOM151CD2HIS2026.59738.15631.9741.0015.96APEPATOM152ND1HIS2027.39238.10229.9411.0015.59APEPATOM153CE1HIS2026.12638.46629.8531.0015.25APEPATOM154NE2HIS2025.62038.50631.0721.0017.34APEPATOM155CHIS2029.67935.81129.9611.0011.56APEPATOM156OHIS2030.78336.05429.4671.009.12APEPATOM157NTHR2128.65035.35529.2601.0012.15APEPATOM158CATHR2128.73935.09027.8281.0012.76APEPATOM159CBTHR2127.34934.68627.2871.0013.90APEPATOM160OG1THR2127.01633.38727.7921.0014.96APEPATOM161CG2THR2127.33634.65825.7561.0013.84APEPATOM162CTHR2129.29436.27827.0251.0012.07APEPATOM163OTHR2130.10236.09026.1111.008.89APEPATOM164NALA2228.87337.49027.3801.0010.72APEPATOM165CAALA2229.31238.69826.6931.0011.63APEPATOM166CBALA2228.31139.81626.9251.0012.20APEPATOM167CALA2230.70639.15627.1021.0013.47APEPATOM168OALA2231.20040.17826.6211.0013.74APEPATOM169NCYS2331.33238.41028.0061.0014.12APEPATOM170CACYS2332.68338.71528.4601.0014.19APEPATOM171CCYS2333.56437.67027.7931.0012.16APEPATOM172OCYS2334.72537.90927.4971.0013.84APEPATOM173CBCYS2332.78238.59929.9951.0012.96APEPATOM174SGCYS2334.45438.85530.6951.0014.19APEPATOM175NLYS2432.98736.50127.5611.0013.18APEPATOM176CALYS2433.69735.40526.9171.0014.00APEPATOM177CBLYS2432.89434.10927.0481.0013.62APEPATOM178CGLYS2433.11133.34728.3341.0013.30APEPATOM179CDLYS2432.59331.92928.1931.0014.90APEPATOM180CELYS2431.65631.54029.3111.0015.48APEPATOM181NZLYS2432.00930.18829.8301.0021.39APEPATOM182CLYS2433.85335.74225.4461.0013.93APEPATOM183OLYS2434.91735.57824.8611.0014.28APEPATOM184NTYR2532.76736.21924.8571.0016.64APEPATOM185CATYR2532.73736.58523.4481.0017.22APEPATOM186CBTYR2531.73635.68422.7191.0018.12APEPATOM187CGTYR2531.71634.24523.2171.0016.13APEPATOM188CD1TYR2530.60033.72723.8791.0018.60APEPATOM189CE1TYR2530.57432.40424.3321.0015.87APEPATOM190CD2TYR2532.81033.40323.0211.0016.98APEPATOM191CE2TYR2532.79432.08123.4691.0014.73APEPATOM192CZTYR2531.67731.59024.1201.0016.64APEPATOM193OHTYR2531.66130.28324.5661.0019.74APEPATOM194CTYR2532.33938.06023.3361.0018.24APEPATOM195OTYR2531.15538.40423.3321.0017.58APEPATOM196NGLY2633.34038.92923.2501.0019.90APEPATOM197CAGLY2633.08640.35823.1821.0022.78APEPATOM198CGLY2632.53640.92721.8861.0025.12APEPATOM199OGLY2632.26042.12521.8151.0026.30APEPATOM200NSER2732.36240.09220.8671.0026.19APEPATOM201CASER2731.85540.57019.5831.0026.72APEPATOM202CBSER2732.96040.43518.5221.0025.95APEPATOM203OGSER2732.45740.04117.2591.0024.78APEPATOM204CSER2730.58639.83919.1391.0026.86APEPATOM205OSER2730.15938.87819.7741.0025.87APEPATOM206NLEU2829.97940.31218.0531.0029.54APEPATOM207CALEU2828.76639.69517.5181.0030.96APEPATOM208CBLEU2827.79340.76917.0211.0033.13APEPATOM209CGLEU2828.12742.21717.3911.0034.56APEPATOM210CD1LEU2829.02242.81216.3191.0034.22APEPATOM211CD2LEU2826.84343.03017.5511.0034.12APEPATOM212CLEU2829.14238.76916.3651.0030.72APEPATOM213OLEU2828.27738.22415.6731.0031.18APEPATOM214NLYS2930.44838.60216.1761.0030.29APEPATOM215CALYS2931.00837.75915.1241.0029.17APEPATOM216CBLYS2932.49038.10214.9371.0031.20APEPATOM217CGLYS2933.01637.86613.5341.0032.99APEPATOM218CDLYS2934.52837.78513.5211.0034.25APEPATOM219CELYS2935.15039.12113.8851.0035.23APEPATOM220NZLYS2935.68639.09815.2731.0037.84APEPATOM221CLYS2930.86736.26915.4441.0027.53APEPATOM222OLYS2931.44635.77216.4131.0025.87APEPATOM223NPRO3030.10435.53014.6211.0027.59APEPATOM224CDPRO3029.36236.01113.4421.0026.03APEPATOM225CAPRO3029.90534.09114.8401.0025.87APEPATOM226CBPRO3028.98233.67513.6941.0025.48APEPATOM227CGPRO3028.33034.94913.2451.0024.87APEPATOM228CPRO3031.18233.25314.8711.0025.41APEPATOM229OPRO3032.06133.40414.0181.0026.47APEPATOM230NASN3131.27332.37615.8661.0022.04APEPATOM231CAASN3132.40731.46916.0301.0021.43APEPATOM232CBASN3133.06131.62317.4131.0021.58APEPATOM233CGASN3133.84032.91117.5641.0023.13APEPATOM234OD1ASN3134.58133.31916.6721.0023.71APEPATOM235ND2ASN3133.68033.55818.7131.0025.47APEPATOM236CASN3131.81730.07115.9441.0019.60APEPATOM237OASN3131.74329.36516.9481.0018.51APEPATOM238NCYS3231.38429.66714.7561.0018.76APEPATOM239CACYS3230.77928.34814.6051.0018.03APEPATOM240CCYS3231.69027.31013.9751.0017.09APEPATOM241OCYS3231.23426.46413.2071.0013.04APEPATOM242CBCYS3229.49328.45613.7921.0017.35APEPATOM243SGCYS3228.25329.52814.5701.0016.28APEPATOM244NGLY3332.97427.37914.3111.0019.59APEPATOM245CAGLY3333.94226.43313.7861.0021.31APEPATOM246CGLY3333.91426.26912.2781.0022.56APEPATOM247OGLY3333.98527.25011.5321.0022.89APEPATOM248NASN3433.81225.02111.8301.0022.35APEPATOM249CAASN3433.78724.72410.4091.0023.03APEPATOM250CBASN3434.53123.41010.1361.0026.79APEPATOM251CGASN3433.75422.18710.5811.0031.53APEPATOM252OD1ASN3433.02822.22111.5791.0035.39APEPATOM253ND2ASN3433.90821.0889.8401.0032.88APEPATOM254CASN3432.37724.6829.8211.0022.38APEPATOM255OASN3432.19324.3518.6471.0021.38APEPATOM256NLYS3531.37725.02910.6291.0019.97APEPATOM257CALYS3530.00725.05310.1331.0017.88APEPATOM258CBLYS3529.01125.16611.2891.0017.85APEPATOM259CGLYS3529.32324.27712.4821.0019.14APEPATOM260CDLYS3528.05023.84713.1791.0018.82APEPATOM261CELYS3528.19623.88414.6891.0018.39APEPATOM262NZLYS3529.49923.32915.1151.0018.61APEPATOM263CLYS3529.87926.2819.2351.0016.90APEPATOM264OLYS3530.55727.2849.4531.0016.79APEPATOM265NVAL3629.02926.2028.2181.0016.21APEPATOM266CAVAL3628.83127.3427.3301.0015.62APEPATOM267CBVAL3628.56026.9165.8721.0015.89APEPATOM268CG1VAL3628.47428.1504.9901.0014.85APEPATOM269CG2VAL3629.66326.0005.3741.0017.84APEPATOM270CVAL3627.63628.1497.8201.0013.37APEPATOM271OVAL3626.53027.6317.9491.0011.50APEPATOM272NVAL3727.88229.4228.0951.0013.37APEPATOM273CAVAL3726.85730.3378.5731.0015.79APEPATOM274CBVAL3727.50631.4509.4241.0016.40APEPATOM275CG1VAL3726.48732.5219.7651.0016.09APEPATOM276CG2VAL3728.09630.84710.6811.0013.21APEPATOM277CVAL3726.06730.9717.4221.0016.67APEPATOM278OVAL3726.55731.8736.7381.0018.09APEPATOM279NVAL3824.84330.4927.2111.0016.92APEPATOM280CAVAL3823.99131.0206.1491.0017.73APEPATOM281CBVAL3822.66230.2296.0511.0015.03APEPATOM282CG1VAL3821.77030.8204.9761.0015.83APEPATOM283CG2VAL3822.95328.7785.7401.0017.06APEPATOM284CVAL3823.70432.4806.4861.0017.90APEPATOM285OVAL3823.85233.3725.6451.0018.01APEPATOM286NSER3923.30532.7137.7311.0015.41APEPATOM287CASER3923.01934.0528.2141.0014.21APEPATOM288CBSER3921.85734.6747.4381.0014.70APEPATOM289OGSER3920.72133.8377.4671.0014.28APEPATOM290CSER3922.67934.0069.7001.0014.75APEPATOM291OSER3922.63632.93610.3081.0012.05APEPATOM292NTYR4022.44435.17910.2781.0014.22APEPATOM293CATYR4022.11135.27211.6861.0014.10APEPATOM294CBTYR4023.39735.17912.5301.0015.42APEPATOM295CGTYR4024.23936.43812.5831.0014.41APEPATOM296CD1TYR4023.92137.47213.4641.0015.34APEPATOM297CE1TYR4024.71138.60513.5631.0016.41APEPATOM298CD2TYR4025.37536.57511.7901.0014.36APEPATOM299CE2TYR4026.17937.71211.8791.0017.60APEPATOM300CZTYR4025.84238.72312.7711.0018.38APEPATOM301OHTYR4026.63939.84112.8961.0019.23APEPATOM302CTYR4021.36036.56911.9691.0013.71APEPATOM303OTYR4021.45637.52611.2011.0013.53APEPATOM304NGLY4120.60236.59013.0611.0012.13APEPATOM305CAGLY4119.85737.78313.4181.0013.27APEPATOM306CGLY4118.38137.65613.1021.0013.46APEPATOM307OGLY4117.96836.72612.4191.0014.55APEPATOM308NLEU4217.58638.60113.5901.0012.94APEPATOM309CALEU4216.15038.58113.3651.0012.38APEPATOM310CBLEU4215.42138.30214.6761.0011.85APEPATOM311CGLEU4215.46236.85815.1701.009.57APEPATOM312CD1LEU4215.27936.82816.6821.0010.07APEPATOM313CD2LEU4214.37436.06314.4751.009.98APEPATOM314CLEU4215.65139.89512.7911.0012.90APEPATOM315OLEU4216.06640.96813.2231.0013.81APEPATOM316NTHR4314.75839.80811.8161.0012.41APEPATOM317CATHR4314.20041.00611.2101.0013.32APEPATOM318CBTHR4313.41240.6939.9191.0011.63APEPATOM319OG1THR4312.19540.02810.2541.0012.20APEPATOM320CG2THR4314.22239.8048.9941.0011.85APEPATOM321CTHR4313.24941.63712.2081.0013.30APEPATOM322OTHR4312.80140.99013.1611.0012.67APEPATOM323NLYS4412.93942.90411.9771.0014.11APEPATOM324CALYS4412.05043.64012.8511.0014.99APEPATOM325CBLYS4411.97545.10012.3791.0016.22APEPATOM326CGLYS4410.59445.66712.1521.0018.80APEPATOM327CDLYS4410.56747.15712.4891.0019.36APEPATOM328CELYS449.65547.91511.5521.0021.90APEPATOM329NZLYS4410.43048.71410.5701.0020.87APEPATOM330CLYS4410.67242.98512.9231.0013.76APEPATOM331OLYS4410.08342.91013.9991.0014.04APEPATOM332NGLN4510.16242.48711.7981.0012.41APEPATOM333CAGLN458.84941.83911.8061.0011.51APEPATOM334CBGLN458.33441.64610.3701.0010.79APEPATOM335CGGLN457.06340.81610.2461.0010.70APEPATOM336CDGLN455.81241.53810.7431.0012.43APEPATOM337OE1GLN455.69642.76310.6501.0012.72APEPATOM338NE2GLN454.86940.77211.2741.0011.44APEPATOM339CGLN458.91740.49612.5481.0010.87APEPATOM340OGLN457.98740.12313.2671.009.48APEPATOM341NGLU4610.02439.77912.3821.009.10APEPATOM342CAGLU4610.20738.49613.0591.009.69APEPATOM343CBGLU4611.51137.84512.6101.008.84APEPATOM344CGGLU4611.36636.91611.4071.009.10APEPATOM345CDGLU4612.71036.53410.8061.009.37APEPATOM346OE1GLU4613.72337.15811.1731.007.41APEPATOM347OE2GLU4612.75535.6079.9661.0010.21APEPATOM348CGLU4610.21738.66614.5821.0010.14APEPATOM349OGLU469.70837.81715.3101.0010.51APEPATOM350NLYS4710.80739.76115.0571.0010.08APEPATOM351CALYS4710.86540.04216.4861.009.91APEPATOM352CBLYS4711.67541.31816.7491.009.24APEPATOM353CGLYS4713.16741.19116.4591.007.94APEPATOM354CDLYS4713.90642.50916.7101.009.13APEPATOM355CELYS4715.41142.36116.4981.0011.45APEPATOM356NZLYS4716.12743.67516.4311.0011.96APEPATOM357CLYS479.43840.22916.9841.0010.20APEPATOM358OLYS479.02739.62617.9691.0010.41APEPATOM359NGLN488.68941.06516.2751.0011.41APEPATOM360CAGLN487.29941.36616.6021.0011.75APEPATOM361CBGLN486.75942.40915.6241.0011.16APEPATOM362CGGLN485.25442.60715.6691.0012.11APEPATOM363CDGLN484.76743.51514.5561.0012.60APEPATOM364OE1GLN485.30144.60614.3591.0010.04APEPATOM365NE2GLN483.75843.06513.8161.0011.92APEPATOM366CGLN486.42040.12316.5631.0012.69APEPATOM367OGLN485.48839.99317.3531.0013.53APEPATOM368NASP496.71639.21915.6331.0012.63APEPATOM369CAASP495.96437.97715.4871.0011.04APEPATOM370CBASP496.29037.32214.1441.0014.99APEPATOM371CGASP495.57837.99012.9811.0017.72APEPATOM372OD1ASP494.51838.62013.2001.0018.74APEPATOM373OD2ASP496.08237.87811.8441.0019.80APEPATOM374CASP496.28536.99816.6151.009.65APEPATOM375OASP495.43336.21117.0201.009.33APEPATOM376NILE507.51937.03417.1071.008.25APEPATOM377CAILE507.91636.15218.2031.008.01APEPATOM378CBILE509.45436.13218.3871.007.72APEPATOM379CG2ILE509.82335.41619.6931.007.19APEPATOM380CG1ILE5010.10335.41017.2031.006.44APEPATOM381CD1ILE5011.58235.68717.0411.004.97APEPATOM382CILE507.25636.62119.4991.008.14APEPATOM383OILE506.80535.80820.3031.007.29APEPATOM384NLEU517.19137.93819.6791.008.57APEPATOM385CALEU516.57138.52920.8541.009.76APEPATOM386CBLEU516.73340.05520.8361.009.57APEPATOM387CGLEU516.50940.84422.1391.0012.08APEPATOM388CD1LEU517.50940.40123.2161.009.69APEPATOM389CD2LEU516.65942.33321.8611.0010.85APEPATOM390CLEU515.09138.17220.8631.0010.95APEPATOM391OLEU514.57137.66421.8611.0012.16APEPATOM392NLYS524.42338.42719.7391.0010.41APEPATOM393CALYS522.99438.15619.6011.0010.29APEPATOM394CBLYS522.52038.53518.1961.0010.94APEPATOM395CGLYS521.06638.95618.1321.0014.35APEPATOM396CDLYS520.25838.02917.2361.0016.34APEPATOM397CELYS52−0.87038.78016.5431.0017.57APEPATOM398NZLYS52−2.10738.81717.3741.0017.77APEPATOM399CLYS522.62736.70919.8931.009.70APEPATOM400OLYS521.55336.43220.4191.009.63APEPATOM401NGLU533.50835.78019.5401.0010.85APEPATOM402CAGLU533.24934.36619.7991.0011.95APEPATOM403CBGLU534.26133.49119.0571.0013.57APEPATOM404CGGLU533.95731.99619.0891.0015.51APEPATOM405CDGLU532.52531.65118.6951.0020.29APEPATOM406OE1GLU531.87632.43917.9711.0021.72APEPATOM407OE2GLU532.04430.57719.1111.0021.86APEPATOM408CGLU533.36234.12021.2941.0012.09APEPATOM409OGLU532.56833.38221.8781.0011.99APEPATOM410NHIS544.35734.75021.9101.0012.11APEPATOM411CAHIS544.58034.61023.3401.0011.34APEPATOM412CBHIS545.82935.39923.7691.009.29APEPATOM413CGHIS547.08934.58423.8171.007.76APEPATOM414CD2HIS547.69533.93324.8401.009.61APEPATOM415ND1HIS547.89534.39422.7161.006.60APEPATOM416CE1HIS548.94133.66323.0561.005.37APEPATOM417NE2HIS548.84433.37024.3401.007.30APEPATOM418CHIS543.36535.14324.0921.0010.89APEPATOM419OHIS542.84434.49224.9831.0010.22APEPATOM420NASN552.91336.33123.7031.0012.43APEPATOM421CAASN551.78436.98224.3561.0013.65APEPATOM422CBASN551.79138.47323.9911.0012.77APEPATOM423CGASN552.95039.23224.6551.0012.31APEPATOM424OD1ASN553.39638.87125.7471.007.53APEPATOM425ND2ASN553.43640.28023.9931.009.40APEPATOM426CASN550.41336.34724.0971.0013.82APEPATOM427OASN55−0.45736.35524.9731.0013.31APEPATOM428NASP560.22135.79522.9071.0012.69APEPATOM429CAASP56−1.03635.13422.5721.0012.59APEPATOM430CBASP56−1.04934.67521.1111.0012.80APEPATOM431CGASP56−1.35935.78720.1431.0013.37APEPATOM432OD1ASP56−1.90236.82520.5651.0013.48APEPATOM433OD2ASP56−1.05935.61518.9451.0015.39APEPATOM434CASP56−1.15333.89923.4501.0010.48APEPATOM435OASP56−2.22433.57723.9531.0010.14APEPATOM436NPHE57−0.04633.19123.6041.009.15APEPATOM437CAPHE57−0.04731.98924.4181.0011.09APEPATOM438CBPHE571.27231.22724.2521.0012.68APEPATOM439CGPHE571.26129.86324.8841.0011.85APEPATOM440CD1PHE570.34628.89924.4711.0012.69APEPATOM441CD2PHE572.15029.54925.9031.0012.28APEPATOM442CE1PHE570.31627.64225.0671.0011.80APEPATOM443CE2PHE572.13228.29626.5081.0012.07APEPATOM444CZPHE571.21627.34226.0921.0012.36APEPATOM445CPHE57−0.26732.34625.8901.0010.66APEPATOM446OPHE57−1.01231.66426.5971.0011.28APEPATOM447NARG580.36033.42326.3491.008.83APEPATOM448CAARG580.20433.83027.7381.0010.25APEPATOM449CBARG581.10735.02428.0571.007.50APEPATOM450CGARG582.48334.61528.5301.007.56APEPATOM451CDARG583.47835.75528.4461.007.29APEPATOM452NEARG583.39136.64929.6011.008.58APEPATOM453CZARG584.02536.45030.7501.007.65APEPATOM454NH1ARG584.79735.38830.9081.008.61APEPATOM455NH2ARG583.89237.31831.7381.009.84APEPATOM456CARG58−1.24634.17028.0321.009.76APEPATOM457OARG58−1.79333.73329.0421.0011.57APEPATOM458NGLN59−1.87434.93227.1411.0010.78APEPATOM459CAGLN59−3.27035.32127.3141.009.25APEPATOM460CBGLN59−3.62136.46426.3681.0011.30APEPATOM461CGGLN59−3.38837.87026.9371.0014.86APEPATOM462CDGLN59−3.25437.92428.4571.0015.40APEPATOM463OE1GLN59−2.30638.50828.9761.0020.39APEPATOM464NE2GLN59−4.20337.32829.1711.0016.19APEPATOM465CGLN59−4.23334.15627.1071.008.85APEPATOM466OGLN59−5.27534.08427.7531.008.65APEPATOM467NLYS60−3.90033.24026.2091.009.28APEPATOM468CALYS60−4.76532.08425.9991.009.29APEPATOM469CBLYS60−4.19031.17024.9191.0011.77APEPATOM470CGLYS60−5.09729.99924.5551.0012.61APEPATOM471CDLYS60−4.35728.67824.6601.0012.86APEPATOM472CELYS60−3.84928.20523.3101.0010.33APEPATOM473NZLYS60−4.53526.97022.8301.0012.74APEPATOM474CLYS60−4.84031.32927.3201.009.51APEPATOM475OLYS60−5.92231.01727.8161.007.72APEPATOM476NILE61−3.67131.04927.8871.009.04APEPATOM477CAILE61−3.57030.34829.1611.009.87APEPATOM478CBILE61−2.07530.10129.5361.0010.78APEPATOM479CG2ILE61−1.97029.59830.9731.0010.13APEPATOM480CG1ILE61−1.42829.15228.5071.009.94APEPATOM481CD1ILE61−1.21227.72028.9801.0010.64APEPATOM482CILE61−4.25431.14130.2831.009.04APEPATOM483OILE61−4.98030.57131.0921.009.20APEPATOM484NALA62−4.04132.45430.3141.008.91APEPATOM485CAALA62−4.62833.30831.3501.009.06APEPATOM486CBALA62−4.09034.72931.2091.005.84APEPATOM487CALA62−6.16533.32731.3631.0011.19APEPATOM488OALA62−6.79433.58132.3971.0012.79APEPATOM489NARG63−6.76933.05330.2141.0012.40APEPATOM490CAARG63−8.21933.05030.0961.0010.93APEPATOM491CBARG63−8.61833.62728.7361.0010.77APEPATOM492CGARG63−8.04335.01228.5051.0012.79APEPATOM493CDARG63−8.60835.68427.2781.0015.66APEPATOM494NEARG63−7.86836.90426.9681.0017.96APEPATOM495CZARG63−7.34637.17925.7771.0020.00APEPATOM496NH1ARG63−7.48336.32124.7721.0019.36APEPATOM497NH2ARG63−6.67938.31325.5901.0022.72APEPATOM498CARG63−8.82731.66130.2851.0010.92APEPATOM499OARG63−10.03631.48930.1791.0012.37APEPATOM500NGLY64−7.98630.67730.5751.0012.22APEPATOM501CAGLY64−8.47529.32530.7801.0011.71APEPATOM502CGLY64−8.98528.68529.5091.0013.26APEPATOM503OGLY64−9.95027.91129.5401.0014.03APEPATOM504NLEU65−8.33128.99828.3911.0011.24APEPATOM505CALEU65−8.71128.46327.0951.0010.84APEPATOM506CBLEU65−8.74729.58126.0441.0010.48APEPATOM507CGLEU65−9.60230.80326.3961.008.01APEPATOM508CD1LEU65−9.27831.94625.4701.0013.03APEPATOM509CD2LEU65−11.07430.45026.2911.0010.77APEPATOM510CLEU65−7.76427.36126.6441.0012.39APEPATOM511OLEU65−7.99826.71925.6251.0012.90APEPATOM512NGLU66−6.68627.14727.3871.0011.27APEPATOM513CAGLU66−5.75426.09427.0231.0012.09APEPATOM514CBGLU66−4.36526.36127.6101.0011.39APEPATOM515CGGLU66−3.32725.30827.2451.0012.91APEPATOM516CDGLU66−3.36224.94125.7741.0013.85APEPATOM517OE1GLU66−2.68925.62924.9881.0018.33APEPATOM518OE2GLU66−4.05423.97125.4011.0012.27APEPATOM519CGLU66−6.32324.79927.5751.0012.38APEPATOM520OGLU66−6.21424.51228.7641.0012.85APEPATOM521NTHR67−6.94324.02226.6961.0013.75APEPATOM522CATHR67−7.55322.76927.0911.0013.16APEPATOM523CBTHR67−8.56222.29526.0181.0013.92APEPATOM524OG1THR67−7.85821.89424.8301.0014.81APEPATOM525CG2THR67−9.52423.41325.6711.0011.42APEPATOM526CTHR67−6.54821.65227.3681.0013.63APEPATOM527OTHR67−6.87520.68228.0491.0014.84APEPATOM528NARG68−5.32621.79326.8611.0013.12APEPATOM529CAARG68−4.30120.77027.0421.0011.47APEPATOM530CBARG68−3.22220.91425.9671.0013.83APEPATOM531CGARG68−3.71520.74124.5381.0013.10APEPATOM532CDARG68−2.62621.12823.5421.0013.89APEPATOM533NEARG68−2.31722.55623.5901.0012.94APEPATOM534CZARG68−1.24423.11123.0331.0011.20APEPATOM535NH1ARG68−0.37222.35122.3831.0010.56APEPATOM536NH2ARG68−1.04224.42023.1351.005.87APEPATOM537CARG68−3.63120.74628.4151.0011.74APEPATOM538OARG68−3.42021.78929.0321.0010.94APEPATOM539NGLY69−3.29519.53628.8671.0011.61APEPATOM540CAGLY69−2.64119.33430.1471.0014.23APEPATOM541CGLY69−2.56517.85730.5181.0016.11APEPATOM542OGLY69−2.99817.00129.7471.0016.74APEPATOM543NASN70−2.00617.55131.6871.0016.09APEPATOM544CAASN70−1.89616.17232.1561.0017.23APEPATOM545CBASN70−0.43915.70432.1321.0018.05APEPATOM546CGASN70−0.31014.20332.2911.0020.68APEPATOM547OD1ASN70−1.20413.45231.8941.0020.26APEPATOM548ND2ASN700.80613.75232.8741.0020.67APEPATOM549CASN70−2.45216.02533.5781.0016.86APEPATOM550OASN70−1.71715.73934.5231.0015.70APEPATOM551NPRO71−3.77016.20433.7381.0016.37APEPATOM552CDPRO71−4.46716.04635.0261.0016.71APEPATOM553CAPRO71−4.71316.52232.6631.0016.24APEPATOM554CBPRO71−5.96215.77733.0861.0016.30APEPATOM555CGPRO71−5.92815.90634.6141.0016.81APEPATOM556CPRO71−4.99918.01232.4911.0016.23APEPATOM557OPRO71−4.63818.83733.3381.0016.06APEPATOM558NGLY72−5.66618.34231.3921.0013.86APEPATOM559CAGLY72−6.04219.72031.1431.0014.67APEPATOM560CGLY72−7.43719.90231.7161.0014.79APEPATOM561OGLY72−8.03018.93532.1921.0016.06APEPATOM562NPRO73−8.00021.11531.6951.0013.41APEPATOM563CDPRO73−9.34321.35432.2531.0013.38APEPATOM564CAPRO73−7.41222.34731.1641.0014.44APEPATOM565CBPRO73−8.62123.25030.9761.0013.74APEPATOM566CGPRO73−9.51922.85032.1131.0012.91APEPATOM567CPRO73−6.41222.97732.1291.0013.52APEPATOM568OPRO73−6.27122.53733.2681.0013.30APEPATOM569NGLN74−5.71324.00431.6581.0012.54APEPATOM570CAGLN74−4.78224.72332.5061.0011.28APEPATOM571CBGLN74−3.70825.43331.6721.0010.31APEPATOM572CGGLN74−2.65824.50531.0431.009.27APEPATOM573CDGLN74−2.07023.48432.0241.0012.07APEPATOM574OE1GLN74−1.55823.83833.0871.0010.98APEPATOM575NE2GLN74−2.13722.21031.6541.0012.44APEPATOM576CGLN74−5.70725.73633.1701.0011.59APEPATOM577OGLN74−6.71026.13932.5791.0012.75APEPATOM578NPRO75−5.39326.15834.4011.0010.76APEPATOM579CDPRO75−4.23025.77035.2211.0010.45APEPATOM580CAPRO75−6.25427.12835.0921.0010.89APEPATOM581CBPRO75−5.87626.94836.5611.0010.78APEPATOM582CGPRO75−4.43026.54036.5151.009.92APEPATOM583CPRO75−6.07728.57134.6421.0010.22APEPATOM584OPRO75−5.01728.95534.1701.0011.44APEPATOM585NPRO76−7.12329.39434.7821.0011.88APEPATOM586CDPRO76−8.46129.10735.3271.0013.85APEPATOM587CAPRO76−6.96830.79134.3651.0013.97APEPATOM588CBPRO76−8.38331.37334.4401.0012.73APEPATOM589CGPRO76−9.28430.24534.8301.0014.35APEPATOM590CPRO76−6.00031.50735.3151.0014.80APEPATOM591OPRO76−5.67230.92736.3821.0014.45APEPATOM592NALA77−5.55332.69734.9301.0015.36APEPATOM593CAALA77−4.61733.45835.7451.0016.81APEPATOM594CBALA77−3.26433.53135.0501.0015.29APEPATOM595CALA77−5.11134.86436.0341.0018.21APEPATOM596OALA77−5.94635.41435.3151.0018.77APEPATOM597NLYS78−4.57835.44737.0951.0019.69APEPATOM598CALYS78−4.94436.79937.4871.0021.45APEPATOM599CBLYS78−5.45336.77938.9341.0019.28APEPATOM600CGLYS78−5.40838.10939.6581.0021.07APEPATOM601CDLYS78−5.93937.96941.0781.0022.93APEPATOM602CELYS78−7.03938.99341.3801.0022.07APEPATOM603NZLYS78−8.41638.44241.1921.0018.83APEPATOM604CLYS78−3.68137.65537.3511.0020.82APEPATOM605OLYS78−3.73538.82536.9731.0022.49APEPATOM606NASN79−2.54637.02437.6321.0020.62APEPATOM607CAASN79−1.22537.65037.5961.0020.96APEPATOM608CBASN79−0.32236.89338.5911.0021.73APEPATOM609CGASN790.89537.69639.0411.0026.41APEPATOM610OD1ASN791.73937.19439.7941.0027.31APEPATOM611ND2ASN790.99538.94138.5861.0030.92APEPATOM612CASN79−0.57037.64936.1991.0020.23APEPATOM613OASN790.65837.67936.1091.0020.51APEPATOM614NMET80−1.35437.64835.1171.0017.31APEPATOM615CAMET80−0.74537.58133.7831.0016.95APEPATOM616CBMET80−1.33436.39833.0141.0014.01APEPATOM617CGMET80−0.47535.94131.8481.0010.95APEPATOM618SDMET801.00135.03232.3601.0010.66APEPATOM619CEMET800.30933.39232.6311.009.65APEPATOM620CMET80−0.74338.80932.8631.0017.13APEPATOM621OMET80−1.78539.23632.3771.0017.76APEPATOM622NLYS810.45039.34332.6021.0016.19APEPATOM623CALYS810.62140.50931.7341.0015.79APEPATOM624CBLYS811.36041.62632.4801.0019.28APEPATOM625CGLYS810.48542.47933.3761.0023.64APEPATOM626CDLYS811.28342.97634.5811.0029.37APEPATOM627CELYS810.65242.55135.9141.0030.79APEPATOM628NZLYS811.63941.85036.7941.0031.43APEPATOM629CLYS811.42840.13530.4891.0015.20APEPATOM630OLYS812.14439.13330.4781.0013.27APEPATOM631NASN821.31740.94729.4451.0014.40APEPATOM632CAASN822.04740.69528.2141.0014.06APEPATOM633CBASN821.44241.49227.0591.0015.84APEPATOM634CGASN820.08140.97026.6361.0019.06APEPATOM635OD1ASN82−0.83741.74626.3661.0020.37APEPATOM636ND2ASN82−0.05839.64926.5791.0020.55APEPATOM637CASN823.49641.10728.4001.0011.87APEPATOM638OASN823.80041.96829.2261.0011.67APEPATOM639NLEU834.38440.48327.6331.0010.14APEPATOM640CALEU835.80940.78927.6841.009.10APEPATOM641CBLEU836.64839.57727.2721.009.30APEPATOM642CGLEU836.37338.23027.9351.009.01APEPATOM643CD1LEU837.07737.11927.1781.0010.20APEPATOM644CD2LEU836.84338.28329.3781.0010.62APEPATOM645CLEU836.10441.91926.7181.007.69APEPATOM646OLEU835.28542.25425.8661.007.01APEPATOM647NVAL847.27742.51626.8781.007.90APEPATOM648CAVAL847.73643.58526.0041.009.09APEPATOM649CBVAL847.88844.94726.7651.009.60APEPATOM650CG1VAL846.51145.47627.1521.0011.40APEPATOM651CG2VAL848.75344.78828.0031.008.85APEPATOM652CVAL849.08843.11025.4791.008.16APEPATOM653OVAL849.72042.24526.0861.007.18APEPATOM654NTRP859.52443.63724.3431.009.08APEPATOM655CATRP8510.80743.22223.8011.008.24APEPATOM656CBTRP8510.84443.41222.2831.007.78APEPATOM657CGTRP8512.05442.78921.6231.007.96APEPATOM658CD2TRP8512.16241.46021.0921.006.06APEPATOM659CE2TRP8513.45941.33020.5441.005.27APEPATOM660CE3TRP8511.29040.36621.0231.005.63APEPATOM661CD1TRP8513.26043.39221.3841.006.75APEPATOM662NE1TRP8514.10442.52220.7371.005.69APEPATOM663CZ2TRP8513.90540.15319.9351.005.15APEPATOM664CZ3TRP8511.73639.19220.4141.003.85APEPATOM665CH2TRP8513.03539.09919.8791.003.87APEPATOM666CTRP8511.92844.01824.4511.009.70APEPATOM667OTRP8511.79045.21424.7131.0012.63APEPATOM668NASN8613.03643.34024.7221.009.17APEPATOM669CAASN8614.19143.98025.3401.008.03APEPATOM670CBASN8614.39943.40726.7481.004.83APEPATOM671CGASN8615.48444.12127.5051.005.77APEPATOM672OD1ASN8616.65743.82627.3321.005.23APEPATOM673ND2ASN8615.10045.07028.3491.006.46APEPATOM674CASN8615.45043.78924.4741.007.66APEPATOM675OASN8615.88542.66724.2151.006.17APEPATOM676NASP8716.02844.89924.0301.008.98APEPATOM677CAASP8717.21344.86623.1791.009.94APEPATOM678CBASP8717.54846.27822.6951.0010.21APEPATOM679CGASP8716.60246.75721.6221.009.93APEPATOM680OD1ASP8716.06545.90220.9011.0010.59APEPATOM681OD2ASP8716.39247.98021.4981.0011.15APEPATOM682CASP8718.44544.24923.8271.0011.13APEPATOM683OASP8719.27143.65123.1411.0011.97APEPATOM684NGLU8818.57644.39525.1421.009.78APEPATOM685CAGLU8819.72843.83625.8381.0010.33APEPATOM686CBGLU8819.84144.42227.2551.0012.21APEPATOM687CGGLU8821.21044.21327.8881.009.98APEPATOM688CDGLU8821.20444.40029.3901.009.88APEPATOM689OE1GLU8820.12544.66029.9571.0013.29APEPATOM690OE2GLU8822.28244.28930.0101.009.90APEPATOM691CGLU8819.66042.31425.9121.009.04APEPATOM692OGLU8820.65141.62925.6581.008.42APEPATOM693NLEU8918.49141.78926.2691.008.21APEPATOM694CALEU8918.30540.34326.3671.007.65APEPATOM695CBLEU8916.88140.01626.8241.007.64APEPATOM696CGLEU8916.49940.39428.2541.006.63APEPATOM697CD1LEU8915.11139.90428.5491.005.53APEPATOM698CD2LEU8917.48739.78529.2371.007.49APEPATOM699CLEU8918.55439.71924.9971.008.92APEPATOM700OLEU8919.21438.68924.8851.007.56APEPATOM701NALA9018.01040.35723.9641.008.77APEPATOM702CAALA9018.16239.90222.5881.009.89APEPATOM703CBALA9017.40640.83021.6541.006.21APEPATOM704CALA9019.64039.84922.1971.009.83APEPATOM705OALA9020.06438.94021.4911.0010.34APEPATOM706NTYR9120.41540.82122.6721.0010.22APEPATOM707CATYR9121.84640.89422.3801.0010.21APEPATOM708CBTYR9122.42642.20322.9211.0011.27APEPATOM709CGTYR9123.92142.32922.7301.0013.72APEPATOM710CD1TYR9124.45842.65321.4871.0014.77APEPATOM711CE1TYR9125.83742.74721.3011.0016.40APEPATOM712CD2TYR9124.80242.10423.7881.0014.30APEPATOM713CE2TYR9126.17842.19523.6141.0014.06APEPATOM714CZTYR9126.68842.51622.3701.0018.00APEPATOM715OHTYR9128.05242.60822.1911.0018.78APEPATOM716CTYR9122.62039.71422.9671.0011.02APEPATOM717OTYR9123.41139.07722.2791.0011.79APEPATOM718NVAL9222.39739.43224.2441.0010.38APEPATOM719CAVAL9223.07538.32524.9031.008.66APEPATOM720CBVAL9222.78538.31926.4271.008.13APEPATOM721CG1VAL9223.48837.14227.0951.005.04APEPATOM722CG2VAL9223.26739.62227.0461.006.97APEPATOM723CVAL9222.63437.00224.2861.009.57APEPATOM724OVAL9223.41836.06324.1941.0010.64APEPATOM725NALA9321.37636.93323.8581.009.31APEPATOM726CAALA9320.85435.72223.2381.009.67APEPATOM727CBALA9319.34935.84823.0301.008.26APEPATOM728CALA9321.56135.48921.8981.009.72APEPATOM729OALA9321.95434.36621.5811.0010.89APEPATOM730NGLN9421.73036.56521.1301.008.57APEPATOM731CAGLN9422.38636.51519.8281.006.19APEPATOM732CBGLN9422.31637.89219.1621.007.13APEPATOM733CGGLN9422.60637.89117.6681.006.55APEPATOM734CDGLN9421.77836.87516.9111.006.86APEPATOM735OE1GLN9420.55137.01816.7751.007.69APEPATOM736NE2GLN9422.44135.83616.4121.004.45APEPATOM737CGLN9423.84336.08219.9461.007.41APEPATOM738OGLN9424.30235.21719.2031.008.55APEPATOM739NVAL9524.57436.69920.8681.007.81APEPATOM740CAVAL9525.97136.35721.0891.006.18APEPATOM741CBVAL9526.55137.11222.3271.008.06APEPATOM742CG1VAL9527.89936.52322.7281.008.13APEPATOM743CG2VAL9526.71638.58322.0111.007.34APEPATOM744CVAL9526.09134.85521.3241.007.07APEPATOM745OVAL9526.94934.19920.7371.003.91APEPATOM746NTRP9625.22434.31222.1801.008.26APEPATOM747CATRP9625.24432.87922.4941.008.88APEPATOM748CBTRP9624.28432.55523.6501.006.54APEPATOM749CGTRP9624.25831.08924.0301.007.96APEPATOM750CD2TRP9625.39030.23224.2401.007.64APEPATOM751CE2TRP9624.89228.94624.5491.007.17APEPATOM752CE3TRP9626.77830.42624.1971.008.39APEPATOM753CD1TRP9623.15030.30524.2171.008.48APEPATOM754NE1TRP9623.52429.01624.5271.005.50APEPATOM755CZ2TRP9625.73427.85924.8121.006.91APEPATOM756CZ3TRP9627.61429.34124.4591.008.97APEPATOM757CH2TRP9627.08728.07624.7611.008.90APEPATOM758CTRP9624.86732.03321.2811.008.85APEPATOM759OTRP9625.50031.01121.0071.008.27APEPATOM760NALA9723.82732.45320.5661.007.57APEPATOM761CAALA9723.39031.72119.3811.009.88APEPATOM762CBALA9722.18232.41518.7421.004.36APEPATOM763CALA9724.54731.66518.3871.008.40APEPATOM764OALA9724.77730.64717.7341.008.69APEPATOM765NASN9825.28232.76718.3001.009.32APEPATOM766CAASN9826.40232.88317.3751.009.40APEPATOM767CBASN9826.89834.33617.3471.008.07APEPATOM768CGASN9826.08435.21716.4021.008.00APEPATOM769OD1ASN9825.09334.77615.8211.0011.11APEPATOM770ND2ASN9826.50036.46416.2501.009.71APEPATOM771CASN9827.56831.92617.6471.009.78APEPATOM772OASN9828.52431.87416.8691.008.97APEPATOM773NGLN9927.49231.16018.7331.008.27APEPATOM774CAGLN9928.55630.21219.0511.009.27APEPATOM775CBGLN9928.77430.12020.5721.0010.68APEPATOM776CGGLN9929.11731.45221.2411.009.08APEPATOM777CDGLN9930.11932.26620.4441.0010.60APEPATOM778OE1GLN9931.19531.78020.1071.0011.69APEPATOM779NE2GLN9929.77233.51120.1461.0012.17APEPATOM780CGLN9928.20528.83918.4841.0010.52APEPATOM781OGLN9929.04927.94218.4261.0011.67APEPATOM782NCYS10026.95928.69018.0471.0011.03APEPATOM783CACYS10026.47427.43917.4701.0012.62APEPATOM784CCYS10026.71126.23418.3731.0014.10APEPATOM785OCYS10027.11325.16617.9061.0013.71APEPATOM786CBCYS10027.12627.18216.1081.0012.51APEPATOM787SGCYS10026.63928.32114.7661.0013.92APEPATOM788NGLN10126.45726.41119.6671.0013.78APEPATOM789CAGLN10126.61525.33720.6401.0014.58APEPATOM790CBGLN10127.65625.72321.6961.0016.78APEPATOM791CGGLN10129.10625.50621.2691.0019.68APEPATOM792CDGLN10130.09726.12522.2391.0020.61APEPATOM793OE1GLN10131.11326.69021.8331.0023.08APEPATOM794NE2GLN10129.80226.02323.5301.0024.57APEPATOM795CGLN10125.27225.09821.3231.0014.56APEPATOM796OGLN10124.98725.71622.3471.0014.71APEPATOM797NTYR10224.45724.20120.7671.0012.66APEPATOM798CATYR10223.13123.91121.3261.0014.54APEPATOM799CBTYR10222.46922.72120.6101.0013.93APEPATOM800CGTYR10221.01522.53121.0121.0013.05APEPATOM801CD1TYR10220.03323.41820.5741.0011.54APEPATOM802CE1TYR10218.71023.29520.9901.0010.81APEPATOM803CD2TYR10220.63221.50521.8811.0013.43APEPATOM804CE2TYR10219.29821.37322.3071.0013.52APEPATOM805CZTYR10218.34822.27621.8531.0011.37APEPATOM806OHTYR10217.03122.15422.2421.0012.72APEPATOM807CTYR10223.12323.63622.8241.0014.75APEPATOM808OTYR10223.82522.74723.3051.0014.15APEPATOM809NGLY10322.30324.39923.5481.0015.34APEPATOM810CAGLY10322.19424.24124.9881.0013.83APEPATOM811CGLY10322.17425.58625.6981.0014.82APEPATOM812OGLY10322.05126.62725.0501.0013.51APEPATOM813NHIS10422.30925.57627.0221.0013.28APEPATOM814CAHIS10422.29326.82127.7921.0013.00APEPATOM815CBHIS10421.53526.62729.1111.0014.84APEPATOM816CGHIS10420.08526.30928.9381.0017.77APEPATOM817CD2HIS10419.34525.26329.3701.0018.90APEPATOM818ND1HIS10419.22427.12528.2361.0019.74APEPATOM819CE1HIS10418.01426.59428.2451.0019.42APEPATOM820NE2HIS10418.06025.46528.9251.0019.61APEPATOM821CHIS10423.68927.32228.1161.0010.17APEPATOM822OHIS10424.57326.53228.4031.008.70APEPATOM823NASP10523.89028.63528.0581.0010.86APEPATOM824CAASP10525.18829.19728.4171.0012.42APEPATOM825CBASP10525.40030.59027.7941.0010.99APEPATOM826CGASP10524.17231.46327.8751.0011.97APEPATOM827OD1ASP10523.05430.91427.9661.0014.08APEPATOM828OD2ASP10524.32432.70527.8441.0011.83APEPATOM829CASP10525.20029.27429.9491.0013.37APEPATOM830OASP10524.14529.25030.5921.0013.12APEPATOM831NTHR10626.39529.36130.5221.0014.55APEPATOM832CATHR10626.57329.38531.9711.0015.70APEPATOM833CBTHR10628.03229.05132.3221.0017.02APEPATOM834OG1THR10628.34927.73931.8371.0019.67APEPATOM835CG2THR10628.24429.10133.8151.0019.92APEPATOM836CTHR10626.18130.66132.7121.0014.86APEPATOM837OTHR10625.64830.59833.8261.0014.67APEPATOM838NCYS10726.44431.81332.1071.0012.86APEPATOM839CACYS10726.13133.08632.7481.0011.94APEPATOM840CCYS10725.60834.11231.7411.0011.28APEPATOM841OCYS10726.35434.59430.8861.008.75APEPATOM842CBCYS10727.38933.61833.4511.0011.80APEPATOM843SGCYS10727.15535.04534.5671.0014.81APEPATOM844NARG10824.32434.44831.8571.0010.42APEPATOM845CAARG10823.69435.40830.9561.0010.25APEPATOM846CBARG10822.65634.70330.0801.007.60APEPATOM847CGARG10821.29934.52530.7461.006.23APEPATOM848CDARG10820.45833.46030.0471.004.46APEPATOM849NEARG10821.06632.13630.1181.008.96APEPATOM850CZARG10820.68831.19230.9711.009.87APEPATOM851NH1ARG10819.70331.42731.8251.009.20APEPATOM852NH2ARG10821.28430.01330.9681.009.69APEPATOM853CARG10823.01536.57531.6671.0010.62APEPATOM854OARG10822.46537.45431.0111.0011.81APEPATOM855NASP10923.05136.58332.9981.0010.19APEPATOM856CAASP10922.42137.64433.7841.009.53APEPATOM857CBASP10922.73737.45735.2671.0011.00APEPATOM858CGASP10922.04936.24835.8641.0010.30APEPATOM859OD1ASP10921.13735.70435.2131.008.82APEPATOM860OD2ASP10922.42035.83936.9841.0012.03APEPATOM861CASP10922.82739.05133.3681.0010.45APEPATOM862OASP10923.93139.27432.8781.0011.18APEPATOM863NVAL11021.91940.00133.5651.0010.47APEPATOM864CAVAL11022.19241.40033.2401.0011.15APEPATOM865CBVAL11021.08342.02532.3461.008.57APEPATOM866CG1VAL11021.28241.60730.8841.008.96APEPATOM867CG2VAL11019.71141.60032.8401.008.45APEPATOM868CVAL11022.26342.16834.5641.0012.34APEPATOM869OVAL11022.04441.59135.6311.0011.18APEPATOM870NALA11122.56743.46034.4931.0012.60APEPATOM871CAALA11122.67044.28335.6911.0014.41APEPATOM872CBALA11123.19245.66535.3291.0014.98APEPATOM873CALA11121.35144.41236.4451.0015.05APEPATOM874OALA11121.34844.49137.6651.0017.05APEPATOM875NLYS11220.23344.42535.7231.0015.22APEPATOM876CALYS11218.91944.56536.3461.0014.82APEPATOM877CBLYS11217.88944.97935.2951.0017.33APEPATOM878CGLYS11216.51845.30135.8541.0018.63APEPATOM879CDLYS11215.72246.15634.8851.0020.78APEPATOM880CELYS11214.27546.29835.3311.0022.40APEPATOM881NZLYS11213.37846.77534.2301.0024.78APEPATOM882CLYS11218.39543.33437.0921.0014.63APEPATOM883OLYS11217.76343.46238.1381.0015.19APEPATOM884NTYR11318.65242.14536.5651.0014.03APEPATOM885CATYR11318.15540.94137.2111.0012.90APEPATOM886CBTYR11316.62740.86637.0621.0014.34APEPATOM887CGTYR11316.09441.27535.7011.0013.96APEPATOM888CD1TYR11316.72540.86734.5291.0014.86APEPATOM889CE1TYR11316.23641.23433.2791.0015.41APEPATOM890CD2TYR11314.95042.06435.5901.0015.52APEPATOM891CE2TYR11314.44742.43934.3451.0017.15APEPATOM892CZTYR11315.09842.02133.1921.0018.32APEPATOM893OHTYR11314.61942.40631.9581.0020.11APEPATOM894CTYR11318.76139.65936.6581.0012.75APEPATOM895OTYR11319.59239.68535.7421.009.39APEPATOM896NGLN11418.33438.54237.2411.0011.10APEPATOM897CAGLN11418.76237.22336.8201.0011.13APEPATOM898CBGLN11418.39736.18237.8721.0013.13APEPATOM899CGGLN11419.49235.92138.8811.0017.82APEPATOM900CDGLN11419.04934.96939.9711.0021.01APEPATOM901OE1GLN11418.98433.75439.7671.0024.17APEPATOM902NE2GLN11418.73535.51741.1401.0021.13APEPATOM903CGLN11417.96936.97735.5491.0010.94APEPATOM904OGLN11416.85137.48935.4181.0010.27APEPATOM905NVAL11518.52936.19534.6261.008.78APEPATOM906CAVAL11517.87935.93633.3391.006.98APEPATOM907CBVAL11518.67936.62832.2041.008.14APEPATOM908CG1VAL11518.03736.35830.8681.009.82APEPATOM909CG2VAL11518.75038.12532.4611.006.98APEPATOM910CVAL11517.66934.45732.9751.006.17APEPATOM911OVAL11518.58133.63433.0931.004.78APEPATOM912NGLY11616.44934.14232.5401.005.97APEPATOM913CAGLY11616.10532.78832.1391.007.14APEPATOM914CGLY11616.36432.56830.6541.007.72APEPATOM915OGLY11616.70633.50429.9301.006.29APEPATOM916NGLN11716.19531.33730.1861.008.62APEPATOM917CAGLN11716.45631.05828.7801.009.69APEPATOM918CBGLN11717.98031.00128.5501.008.96APEPATOM919CGGLN11718.41930.46527.1791.008.07APEPATOM920CDGLN11719.93530.33127.0461.007.90APEPATOM921OE1GLN11720.50729.28827.3601.0010.49APEPATOM922NE2GLN11720.58631.38626.5751.007.02APEPATOM923CGLN11715.81329.79028.2301.009.46APEPATOM924OGLN11715.71328.77528.9201.008.69APEPATOM925NASN11815.37229.87626.9781.0010.04APEPATOM926CAASN11814.76228.75926.2531.009.76APEPATOM927CBASN11813.28029.03725.9501.009.04APEPATOM928CGASN11812.35728.73527.1271.009.64APEPATOM929OD1ASN11812.69627.97628.0351.009.37APEPATOM930ND2ASN11811.17829.33727.1081.008.63APEPATOM931CASN11815.52628.67424.9261.009.88APEPATOM932OASN11815.84729.70724.3421.009.07APEPATOM933NVAL11915.83627.46424.4651.0010.24APEPATOM934CAVAL11916.53327.28823.1881.009.82APEPATOM935CBVAL11918.02126.81123.3401.009.70APEPATOM936CG1VAL11918.76427.68424.3491.0011.24APEPATOM937CG2VAL11918.07225.34423.7491.0011.10APEPATOM938CVAL11915.78426.24722.3791.0010.42APEPATOM939OVAL11915.11625.38022.9391.007.84APEPATOM940NALA12015.89426.34521.0571.0011.69APEPATOM941CAALA12015.22425.41620.1641.0010.58APEPATOM942CBALA12013.85325.96019.7831.009.38APEPATOM943CALA12016.06525.20318.9131.0011.91APEPATOM944OALA12016.74926.11418.4471.0011.05APEPATOM945NLEU12116.00523.99918.3631.0011.80APEPATOM946CALEU12116.76223.70717.1641.0010.62APEPATOM947CBLEU12118.21923.42317.5341.0012.00APEPATOM948CGLEU12119.16223.06516.3831.0014.58APEPATOM949CD1LEU12119.91424.31015.9371.0014.77APEPATOM950CD2LEU12120.12421.97516.8301.0016.44APEPATOM951CLEU12116.19022.52116.3951.0011.28APEPATOM952OLEU12115.74421.54016.9891.008.33APEPATOM953NTHR12216.18322.63315.0691.009.47APEPATOM954CATHR12215.72321.55114.2031.009.80APEPATOM955CBTHR12214.28221.76613.6911.008.73APEPATOM956OG1THR12214.27222.80112.7041.008.66APEPATOM957CG2THR12213.35722.13314.8381.0011.47APEPATOM958CTHR12216.66621.50213.0091.009.64APEPATOM959OTHR12217.23222.52412.6161.009.15APEPATOM960NGLY12316.84720.30812.4511.009.74APEPATOM961CAGLY12317.72820.13711.3131.008.54APEPATOM962CGLY12317.04819.32610.2281.008.95APEPATOM963OGLY12316.19918.48210.5141.009.03APEPATOM964NSER12417.42019.5808.9791.007.43APEPATOM965CASER12416.82418.8747.8571.009.14APEPATOM966CBSER12415.58419.6427.3931.0010.09APEPATOM967OGSER12415.33319.4596.0161.0011.96APEPATOM968CSER12417.82718.7186.7091.009.54APEPATOM969OSER12418.71619.5516.5371.0010.56APEPATOM970NTHR12517.69317.6415.9361.0010.06APEPATOM971CATHR12518.59117.4154.8121.0010.19APEPATOM972CBTHR12518.51315.9744.2571.0011.20APEPATOM973OG1THR12517.14215.5934.0861.0012.88APEPATOM974CG2THR12519.21815.0015.1911.008.70APEPATOM975CTHR12518.27418.3693.6761.009.96APEPATOM976OTHR12519.08118.5322.7721.0010.12APEPATOM977NALA12617.10318.9993.7311.0010.40APEPATOM978CAALA12616.67819.9552.7051.0011.31APEPATOM979CBALA12615.16919.8632.4921.0011.00APEPATOM980CALA12617.06021.3833.0861.0013.19APEPATOM981OALA12617.11621.7354.2711.0012.55APEPATOM982NALA12717.31422.2072.0781.0012.78APEPATOM983CAALA12717.70023.5902.3151.0015.72APEPATOM984CBALA12718.47124.1351.1061.0015.36APEPATOM985CALA12716.49624.4742.6101.0017.23APEPATOM986OALA12716.08025.2711.7731.0017.44APEPATOM987NLYS12815.94124.3243.8101.0019.74APEPATOM988CALYS12814.79025.1104.2511.0019.25APEPATOM989CBLYS12813.48124.3873.9171.0021.28APEPATOM990CGLYS12812.93024.7212.5271.0026.82APEPATOM991CDLYS12812.08325.9932.5491.0027.74APEPATOM992CELYS12811.58226.3651.1521.0027.56APEPATOM993NZLYS12810.37627.2581.1911.0024.53APEPATOM994CLYS12814.91825.3115.7601.0020.56APEPATOM995OLYS12815.29924.3846.4881.0018.95APEPATOM996NTYR12914.59926.5176.2241.0019.02APEPATOM997CATYR12914.71226.8537.6441.0018.90APEPATOM998CBTYR12915.72827.9857.8121.0017.17APEPATOM999CGTYR12917.06027.6457.1881.0015.78APEPATOM1000CD1TYR12917.31927.9345.8471.0015.45APEPATOM1001CE1TYR12918.51927.5645.2501.0013.12APEPATOM1002CD2TYR12918.04326.9847.9181.0016.01APEPATOM1003CE2TYR12919.25026.6107.3301.0016.66APEPATOM1004CZTYR12919.47926.9005.9941.0015.93APEPATOM1005OHTYR12920.65226.4955.4041.0011.93APEPATOM1006CTYR12913.38427.2138.3121.0018.94APEPATOM1007OTYR12912.57427.9807.7751.0019.85APEPATOM1008NASP13013.17826.6459.4961.0017.23APEPATOM1009CAASP13011.95326.84410.2591.0016.64APEPATOM1010CBASP13012.01226.05011.5681.0018.70APEPATOM1011CGASP13011.39924.67711.4461.0018.76APEPATOM1012OD1ASP13011.06724.26710.3191.0017.38APEPATOM1013OD2ASP13011.25324.00512.4891.0020.96APEPATOM1014CASP13011.61528.28510.5881.0014.91APEPATOM1015OASP13012.48929.11110.8311.0014.08APEPATOM1016NASP13110.31728.55710.5841.0016.25APEPATOM1017CAASP1319.75929.85810.9111.0016.60APEPATOM1018CBASP1318.25529.83410.5711.0019.29APEPATOM1019CGASP1317.55831.16610.8071.0024.36APEPATOM1020OD1ASP1318.03631.97811.6301.0027.58APEPATOM1021OD2ASP1316.50631.39610.1681.0028.19APEPATOM1022CASP1319.99329.96112.4281.0015.04APEPATOM1023OASP1319.70829.01213.1591.0014.34APEPATOM1024NPRO13210.53431.09212.9101.0012.77APEPATOM1025CDPRO13210.95032.27612.1391.0012.42APEPATOM1026CAPRO13210.78831.25314.3541.0013.80APEPATOM1027CBPRO13211.19732.72214.4921.0013.81APEPATOM1028CGPRO13211.71133.10413.1491.0014.43APEPATOM1029CPRO1329.59230.89515.2511.0013.34APEPATOM1030OPRO1329.75830.23916.2781.0012.55APEPATOM1031NVAL1338.39631.32514.8501.0012.80APEPATOM1032CAVAL1337.17031.05415.5911.0012.13APEPATOM1033CBVAL1335.94431.66114.8621.0011.80APEPATOM1034CG1VAL1334.65331.03215.3641.0010.96APEPATOM1035CG2VAL1335.92333.15915.0641.0013.44APEPATOM1036CVAL1336.96429.54915.7441.0013.10APEPATOM1037OVAL1336.45229.08316.7631.0011.95APEPATOM1038NLYS1347.36128.79614.7211.0013.08APEPATOM1039CALYS1347.22727.34114.7381.0014.09APEPATOM1040CBLYS1347.59426.75613.3741.0014.78APEPATOM1041CGLYS1347.71625.23813.3671.0017.92APEPATOM1042CDLYS1347.27324.66112.0241.0020.75APEPATOM1043CELYS1347.45423.14711.9741.0021.88APEPATOM1044NZLYS1347.97922.70410.6461.0022.20APEPATOM1045CLYS1348.12526.73415.8051.0013.26APEPATOM1046OLYS1347.77525.73216.4371.0012.31APEPATOM1047NLEU1359.28927.34315.9901.0012.25APEPATOM1048CALEU13510.24526.88316.9871.0012.29APEPATOM1049CBLEU13511.60427.55116.7551.0011.92APEPATOM1050CGLEU13512.37126.96815.5631.0012.40APEPATOM1051CD1LEU13513.67327.70315.3541.0010.11APEPATOM1052CD2LEU13512.63325.49215.8161.0013.69APEPATOM1053CLEU1359.71127.22218.3711.0011.79APEPATOM1054OLEU1359.86226.44319.3111.0012.52APEPATOM1055NVAL1369.07028.37818.4921.0011.76APEPATOM1056CAVAL1368.50728.80519.7731.0011.55APEPATOM1057CBVAL1367.92630.23619.6741.009.15APEPATOM1058CG1VAL1367.04330.53120.8741.009.61APEPATOM1059CG2VAL1369.05331.24719.5871.005.53APEPATOM1060CVAL1367.40527.83620.2271.0012.73APEPATOM1061OVAL1367.37027.42121.3851.0011.68APEPATOM1062NLYS1376.52127.47719.2981.0013.14APEPATOM1063CALYS1375.42226.55419.5631.0012.46APEPATOM1064CBLYS1374.56226.37618.3071.0012.12APEPATOM1065CGLYS1373.86627.65917.8471.0016.24APEPATOM1066CDLYS1372.76327.39116.8361.0013.34APEPATOM1067CELYS1371.56028.29717.0641.0016.29APEPATOM1068NZLYS1370.43327.56517.7061.0012.20APEPATOM1069CLYS1375.93925.19820.0311.0012.60APEPATOM1070OLYS1375.18324.40620.5891.0013.30APEPATOM1071NMET1387.22024.92419.7971.0012.70APEPATOM1072CAMET1387.80723.66220.2401.0015.80APEPATOM1073CBMET1389.26623.54519.7791.0017.09APEPATOM1074CGMET1389.47822.76718.4821.0021.36APEPATOM1075SDMET13811.11123.08917.7111.0026.01APEPATOM1076CEMET13812.06621.67318.2721.0023.00APEPATOM1077CMET1387.75523.66521.7681.0015.35APEPATOM1078OMET1387.44722.65022.3951.0015.78APEPATOM1079NTRP1398.06924.82422.3461.0013.56APEPATOM1080CATRP1398.06925.03523.7911.0010.21APEPATOM1081CBTRP1398.70026.39524.1221.006.88APEPATOM1082CGTRP13910.11226.58223.5891.007.62APEPATOM1083CD2TRP13910.74627.82123.2201.004.45APEPATOM1084CE2TRP13912.05127.50722.7841.004.04APEPATOM1085CE3TRP13910.33529.16023.2141.004.82APEPATOM1086CD1TRP13911.03725.60623.3671.006.61APEPATOM1087NE1TRP13912.20326.15122.8861.005.06APEPATOM1088CZ2TRP13912.95528.49022.3471.002.98APEPATOM1089CZ3TRP13911.22930.13722.7781.002.00APEPATOM1090CH2TRP13912.52529.79522.3511.004.18APEPATOM1091CTRP1396.62824.99724.3121.0011.53APEPATOM1092OTRP1396.35024.41925.3651.0011.91APEPATOM1093NGLU1405.72325.62223.5571.0011.23APEPATOM1094CAGLU1404.30625.69023.8901.0010.95APEPATOM1095CBGLU1403.53826.42722.7981.009.60APEPATOM1096CGGLU1403.62227.91922.8341.007.49APEPATOM1097CDGLU1402.89328.54421.6661.008.80APEPATOM1098OE1GLU1401.93727.92121.1501.0011.85APEPATOM1099OE2GLU1403.27729.65421.2591.0012.42APEPATOM1100CGLU1403.67224.32124.0381.0011.~5APEPATOM1101OGLU1402.89124.08924.9601.0013.93APEPATOM1102NASP1413.99323.42323.1121.0012.05APEPATOM1103CAASP1413.43322.07823.1061.0013.22APEPATOM1104CBASP1413.85021.34621.8331.0012.72APEPATOM1105CGASP1413.20021.92320.6011.0013.07APEPATOM1106OD1ASP1412.24022.70620.7471.0012.10APEPATOM1107OD2ASP1413.64621.59919.4841.0016.74APEPATOM1108CASP1413.78221.23524.3201.0013.83APEPATOM1109OASP1413.19920.17224.5301.0013.83APEPATOM1110NGLU1424.72621.70525.1241.0014.37APEPATOM1111CAGLU1425.11020.97426.3231.0013.86APEPATOM1112CBGLU1426.33521.62626.9741.0013.22APEPATOM1113CGGLU1427.61921.44926.1581.0013.31APEPATOM1114CDGLU1428.86621.89626.8891.0011.68APEPATOM1115OE1GLU1428.74922.70627.8291.0014.70APEPATOM1116OE2GLU1429.96821.43926.5231.0010.37APEPATOM1117CGLU1423.93720.95727.3011.0014.65APEPATOM1118OGLU1423.81920.04928.1201.0016.37APEPATOM1119NVAL1433.06321.95427.1971.0014.52APEPATOM1120CAVAL1431.90422.07128.0841.0014.56APEPATOM1121CBVAL1430.97023.22127.6441.0014.44APEPATOM1122CG1VAL1430.21022.83426.3761.0012.33APEPATOM1123CG2VAL143−0.00523.54828.7691.0010.75APEPATOM1124CVAL1431.05720.80928.2371.0016.78APEPATOM1125OVAL1430.39920.63129.2581.0016.28APEPATOM1126NLYS1441.05919.94227.2281.0017.87APEPATOM1127CALYS1440.28118.70627.2931.0019.21APEPATOM1128CBLYS1440.25718.02525.9131.0021.23APEPATOM1129CGLYS1441.60217.47125.4521.0023.20APEPATOM1130CDLYS1441.57815.94925.3461.0025.43APEPATOM1131CELYS1442.73915.42324.5061.0025.49APEPATOM1132NZLYS1442.96016.24423.2821.0024.96APEPATOM1133CLYS1440.85217.74628.3501.0018.76APEPATOM1134OLYS1440.18816.79428.7761.0018.94APEPATOM1135NASP1452.08018.00828.7741.0017.39APEPATOM1136CAASP1452.74317.18029.7781.0018.35APEPATOM1137CBASP1454.19916.91629.3641.0020.30APEPATOM1138CGASP1454.31615.94228.1951.0020.35APEPATOM1139OD1ASP1453.37415.15327.9591.0022.15APEPATOM1140OD2ASP1455.35915.96627.5101.0021.64APEPATOM1141CASP1452.71417.82931.1731.0017.37APEPATOM1142OASP1453.06917.19232.1641.0015.03APEPATOM1143NTYR1462.28419.09031.2401.0017.14APEPATOM1144CATYR1462.20019.82132.5061.0015.39APEPATOM1145CBTYR1462.36821.32032.2641.0014.76APEPATOM1146CGTYR1462.69622.07133.5331.0015.28APEPATOM1147CD1TYR1463.99222.05334.0651.0014.62APEPATOM1148CE1TYR1464.28622.68235.2771.0013.76APEPATOM1149CD2TYR1461.70522.74334.2421.0014.99APEPATOM1150CE2TYR1461.99123.37735.4571.0013.01APEPATOM1151CZTYR1463.28123.34035.9641.0012.87APEPATOM1152OHTYR1463.56323.95837.1621.0012.82APEPATOM1153CTYR1460.90619.58033.2941.0016.31APEPATOM1154OTYR146−0.20219.83732.8041.0015.92APEPATOM1155NASN1471.06219.09734.5251.0015.90APEPATOM1156CAASN147−0.06618.79235.4151.0017.53APEPATOM1157CBASN1470.26517.55136.2481.0017.07APEPATOM1158CGASN147−0.85117.17237.1931.0018.14APEPATOM1159OD1ASN147−1.88517.83537.2421.0019.58APEPATOM1160ND2ASN147−0.65116.09637.9491.0015.35APEPATOM1161CASN147−0.40519.95736.3551.0017.15APEPATOM1162OASN1470.28920.19637.3341.0016.44APEPATOM1163NPRO148−1.49920.67736.0821.0017.84APEPATOM1164CDPRO148−2.46220.50834.9811.0016.74APEPATOM1165CAPRO148−1.85421.80536.9511.0019.69APEPATOM1166CBPRO148−2.98222.50436.1891.0017.29APEPATOM1167CGPRO148−3.58821.43635.3671.0017.85APEPATOM1168CPRO148−2.24621.46738.3951.0021.26APEPATOM1169OPRO148−2.04022.28539.2891.0022.85APEPATOM1170NLYS149−2.80220.27538.6241.0023.35APEPATOM1171CALYS149−3.21919.86239.9701.0025.52APEPATOM1172CBLYS149−4.00618.54639.9171.0025.99APEPATOM1173CGLYS149−5.03618.47738.8001.0029.64APEPATOM1174CDLYS149−6.43818.80039.3071.0030.49APEPATOM1175CELYS149−7.16219.77538.3821.0030.51APEPATOM1176NZLYS149−8.40120.35139.0041.0030.17APEPATOM1177CLYS149−2.04119.70440.9291.0026.61APEPATOM1178OLYS149−2.15319.08241.9931.0027.31APEPATOM1179NLYS150−0.90520.27140.5591.0026.69APEPATOM1180CALYS1500.26220.18641.4081.0027.83APEPATOM1181CBLYS1500.90418.79541.2601.0026.22APEPATOM1182CGLYS1502.20518.71840.4951.0025.69APEPATOM1183CDLYS1502.41617.32039.9081.0025.52APEPATOM1184CELYS1502.14016.21440.9221.0024.65APEPATOM1185NZLYS1500.69515.84740.9711.0023.30APEPATOM1186CLYS1501.21821.32041.0621.0029.22APEPATOM1187OLYS1501.50421.57739.8951.0031.30APEPATOM1188NLYS1511.68122.02342.0881.0030.63APEPATOM1189CALYS1512.58123.14841.8961.0029.98APEPATOM1190CBLYS1513.13423.62243.2441.0030.75APEPATOM1191CGLYS1512.30824.73843.8881.0032.85APEPATOM1192CDLYS1512.60526.09343.2461.0032.11APEPATOM1193CELYS1511.51227.10443.5621.0030.61APEPATOM1194NZLYS1512.06128.33144.1961.0027.07APEPATOM1195CLYS1513.72022.80140.9561.0028.42APEPATOM1196OLYS1513.98421.63340.6851.0028.09APEPATOM1197NPHE1524.37723.84240.4601.0028.09APEPATOM1198CAPHE1525.49423.71939.5391.0027.23APEPATOM1199CBPHE1526.13825.09839.3491.0023.04APEPATOM1200CGPHE1527.48625.06438.6871.0021.73APEPATOM1201CD1PHE1527.59524.95137.3071.0019.77APEPATOM1202CD2PHE1528.64625.17139.4421.0021.08APEPATOM1203CE1PHE1528.83324.94836.6881.0018.18APEPATOM1204CE2PHE1529.89425.16938.8321.0020.33APEPATOM1205CZPHE1529.98625.05737.4471.0020.45APEPATOM1206CPHE1526.54322.70839.9961.0028.98APEPATOM1207OPHE1526.82121.73639.2931.0029.79APEPATOM1208NSER1537.11222.94041.1761.0030.67APEPATOM1209CASER1538.16222.08441.7351.0032.34APEPATOM1210CBSER1538.31322.35743.2341.0033.66APEPATOM1211OGSER1539.53921.83443.7131.0035.63APEPATOM1212CSER1537.99020.58141.5221.0031.88APEPATOM1213OSER1538.97719.85941.3421.0029.52APEPATOM1214NGLY1546.74420.11341.5471.0032.09APEPATOM1215CAGLY1546.48818.69441.3731.0031.42APEPATOM1216CGLY1546.12418.28239.9621.0031.52APEPATOM1217OGLY1545.31717.37139.7711.0031.82APEPATOM1218NASN1556.71918.94138.9731.0030.65APEPATOM1219CAASN1556.44818.63537.5731.0028.76APEPATOM1220CBASN1555.79619.84236.8931.0027.64APEPATOM1221CGASN1554.33219.61436.5791.0026.54APEPATOM1222OD1ASN1553.99118.87335.6521.0025.37APEPATOM1223ND2ASN1553.45520.24837.3541.0023.70APEPATOM1224CASN1557.72918.25736.8331.0029.00APEPATOM1225OASN1558.82818.63637.2421.0028.36APEPATOM1226NASP1567.58017.50735.7441.0028.55APEPATOM1227CAASP1568.71417.07134.9331.0028.23APEPATOM1228CBASP1568.21916.10833.8481.0029.34APEPATOM1229CGASP1569.25115.05833.4741.0030.85APEPATOM1230OD1ASP1568.91513.85533.5151.0030.64APEPATOM1231OD2ASP15610.39615.43433.1331.0032.06APEPATOM1232CASP1569.39918.28134.2841.0027.76APEPATOM1233OASP1568.97118.74633.2301.0027.49APEPATOM1234NPHE15710.46418.78934.8971.0028.19APEPATOM1235CAPHE15711.12919.95134.3261.0029.10APEPATOM1236CBPHE15711.96320.70535.3871.0032.07APEPATOM1237CGPHE15713.06219.89336.0381.0035.87APEPATOM1238CD1PHE15714.22619.55535.3301.0036.99APEPATOM1239CD2PHE15712.98219.55537.3971.0035.92APEPATOM1240CE1PHE15715.29718.90135.9661.0036.12APEPATOM1241CE2PHE15714.04718.90238.0441.0036.27APEPATOM1242CZPHE15715.20818.57737.3231.0036.25APEPATOM1243CPHE15711.96719.63933.1001.0028.86APEPATOM1244OPHE15712.42320.54332.4001.0028.11APEPATOM1245NLEU15812.15818.35732.8241.0028.54APEPATOM1246CALEU15812.92817.96231.6531.0028.65APEPATOM1247CBLEU15813.68516.65731.9221.0029.80APEPATOM1248CGLEU15815.21716.66031.8421.0028.78APEPATOM1249CD1LEU15815.68815.22331.6921.0029.48APEPATOM1250CD2LEU15815.70617.50730.6691.0026.31APEPATOM1251CLEU15811.96217.77330.4881.0027.95APEPATOM1252OLEU15812.37517.50129.3661.0030.04APEPATOM1253NLYS15910.67117.93230.7631.0026.29APEPATOM1254CALYS1599.65417.77429.7341.0024.09APEPATOM1255CBLYS1598.80116.54230.0391.0023.00APEPATOM1256CGLYS1599.61915.26530.2031.0024.44APEPATOM1257CDLYS1598.74914.03530.4031.0023.01APEPATOM1258CELYS1597.41414.15429.6911.0022.17APEPATOM1259NZLYS1596.36313.36230.3841.0021.65APEPATOM1260CLYS1598.75619.00029.5951.0022.82APEPATOM1261OLYS1598.09919.18228.5661.0020.81APEPATOM1262NTHR1608.73119.84530.6231.0021.93APEPATOM1263CATHR1607.88921.04130.5901.0019.90APEPATOM1264CBTHR1606.68420.88431.5541.0017.71APEPATOM1265OG1THR1607.16320.72132.8941.0017.45APEPATOM1266CG2THR1605.85619.67031.1821.0014.19APEPATOM1267CTHR1608.61922.35230.9211.0019.16APEPATOM1268OTHR1608.00523.41930.9371.0020.80APEPATOM1269NGLY1619.92522.27031.1601.0017.07APEPATOM1270CAGLY16110.70723.44631.5061.0015.84APEPATOM1271CGLY16110.62924.67930.6161.0015.32APEPATOM1272OGLY16110.82525.79631.0931.0015.24APEPATOM1273NHIS16210.35624.49829.3291.0015.82APEPATOM1274CAHIS16210.27425.63728.4211.0014.73APEPATOM1275CBHIS16210.58725.19326.9951.0017.02APEPATOM1276CGHIS16211.97924.67526.8231.0020.85APEPATOM1277CD2HIS16213.16225.12027.3081.0021.78APEPATOM1278ND1HIS16212.26823.55426.0761.0023.37APEPATOM1279CE1HIS16213.57223.33326.1071.0024.65APEPATOM1280NE2HIS16214.13624.26926.8481.0024.25APEPATOM1281CHIS1628.89326.27728.4861.0013.27APEPATOM1282OHIS1628.75327.49728.4131.0011.88APEPATOM1283NTYR1637.87525.44228.6281.0011.72APEPATOM1284CATYR1636.50925.92628.7331.0011.23APEPATOM1285CBTYR1635.55024.75028.9241.0010.40APEPATOM1286CGTYR1634.27125.12229.6531.0010.24APEPATOM1287CD1TYR1633.36926.02829.0951.0010.55APEPATOM1288CE1TYR1632.19626.37829.7591.009.30APEPATOM1289CD2TYR1633.97024.57630.8981.006.58APEPATOM1290CE2TYR1632.80224.92031.5711.008.87APEPATOM1291CZTYR1631.91825.81830.9951.0010.63APEPATOM1292OHTYR1630.74526.14331.6351.0010.74APEPATOM1293CTYR1636.38726.86329.9371.0010.06APEPATOM1294OTYR1635.91927.99429.8201.0010.02APEPATOM1295NTHR1646.83026.38231.0931.009.35APEPATOM1296CATHR1646.74027.14332.3351.008.02APEPATOM1297CBTHR1647.25926.30133.5111.006.41APEPATOM1298OG1THR1648.57125.81133.2091.005.81APEPATOM1299CG2THR1646.31225.11133.7501.002.00APEPATOM1300CTHR1647.42228.51332.3271.008.62APEPATOM1301OTHR1646.96229.43333.0101.008.07APEPATOM1302NGLN1658.50828.66331.5701.008.53APEPATOM1303CAGLN1659.18329.96431.4991.007.81APEPATOM1304CBGLN16510.59829.83730.9301.007.00APEPATOM1305CGGLN16511.24131.17930.6041.007.27APEPATOM1306CDGLN16511.53732.02331.8401.007.94APEPATOM1307OE1GLN16512.63131.96332.4071.007.98APEPATOM1308NE2GLN16510.56632.81532.2571.005.43APEPATOM1309CGLN1658.37030.90230.6091.007.95APEPATOM1310OGLN1658.36332.11330.8111.008.38APEPATOM1311NMET1667.68330.33029.6251.007.26APEPATOM1312CAMET1666.85931.11828.7151.008.35APEPATOM1313CBMET1666.37730.25327.5531.007.91APEPATOM1314CGMET1667.24530.35626.3091.007.80APEPATOM1315SDMET1666.48629.50024.9311.0012.38APEPATOM1316CEMET1665.50830.82324.2071.0011.75APEPATOM1317CMET1665.65431.74629.4091.006.47APEPATOM1318OMET1665.31332.89029.1281.007.34APEPATOM1319NVAL1675.01131.00930.3141.007.21APEPATOM1320CAVAL1673.84731.55031.0181.007.06APEPATOM1321CBVAL1672.67630.52631.0651.007.32APEPATOM1322CG1VAL1672.29530.10729.6541.003.76APEPATOM1323CG2VAL1673.04829.32131.9011.005.77APEPATOM1324CVAL1674.12532.04632.4441.008.39APEPATOM1325OVAL1673.20032.21133.2311.008.06APEPATOM1326NTRP1685.39632.29032.7671.009.11APEPATOM1327CATRP1685.79332.78434.0891.008.50APEPATOM1328CBTRP1687.32032.74534.2321.007.79APEPATOM1329CGTRP1687.80632.69035.6571.009.67APEPATOM1330CD2TRP1687.96031.51936.4741.008.72APEPATOM1331CE2TRP1688.45231.94637.7251.0010.18APEPATOM1332CE3TRP1687.73030.15336.2681.0010.13APEPATOM1333CD1TRP1688.20033.74736.4301.008.39APEPATOM1334NE1TRP1688.58933.30937.6701.008.61APEPATOM1335CZ2TRP1688.72231.05438.7691.008.53APEPATOM1336CZ3TRP1687.99829.26537.3051.006.40APEPATOM1337CH2TRP1688.48829.72138.5391.009.27APEPATOM1338CTRP1685.29434.21334.2751.008.27APEPATOM1339OTRP1685.78235.13633.6271.008.24APEPATOM1340NALA1694.32434.39235.1671.007.85APEPATOM1341CAALA1693.73435.70435.4151.007.42APEPATOM1342CBALA1692.66835.58536.4761.006.86APEPATOM1343CALA1694.71536.80535.7971.009.10APEPATOM1344OALA1694.52537.96835.4331.0010.62APEPATOM1345NASN1705.75836.43636.5311.009.55APEPATOM1346CAASN1706.76037.39236.9901.0010.81APEPATOM1347CBASN1707.55736.79238.1581.0010.24APEPATOM1348CGASN1706.90737.05739.5131.0011.09APEPATOM1349OD1ASN1705.75837.49739.5981.0011.64APEPATOM1350ND2ASN1707.64336.78340.5781.0014.04APEPATOM1351CASN1707.71637.85935.8871.0010.96APEPATOM1352OASN1708.31738.91835.9991.0010.88APEPATOM1353NTHR1717.86637.07134.8301.0010.69APEPATOM1354CATHR1718.74137.47233.7331.009.90APEPATOM1355CBTHR1719.00236.30832.7571.008.82APEPATOM1356OG1THR1719.73835.27833.4301.0010.00APEPATOM1357CG2THR1719.79336.79031.5411.006.32APEPATOM1358CTHR1718.02638.59232.9921.0010.43APEPATOM1359OTHR1716.84238.46832.6691.0011.58APEPATOM1360NLYS1728.73639.68032.7151.0011.90APEPATOM1361CALYS1728.13140.81832.0271.0012.07APEPATOM1362CBLYS1728.17642.05432.9341.0013.23APEPATOM1363CGLYS1727.42441.89334.2611.0016.65APEPATOM1364CDLYS1725.90541.83034.0741.0018.48APEPATOM1365CELYS1725.35443.04433.3121.0021.38APEPATOM1366NZLYS1724.38643.85234.1171.0019.33APEPATOM1367CLYS1728.75141.16630.6771.009.74APEPATOM1368OLYS1728.12241.83429.8561.009.87APEPATOM1369NGLU1739.98240.71930.4501.0011.94APEPATOM1370CAGLU17310.68441.00529.1981.0013.45APEPATOM1371CBGLU17311.81242.01429.4381.0016.52APEPATOM1372CGGLU17311.75242.72130.7781.0021.67APEPATOM1373CDGLU17311.69544.22030.6181.0023.96APEPATOM1374OE1GLU17311.72744.67929.4551.0024.88APEPATOM1375OE2GLU17311.62144.93531.6431.0028.83APEPATOM1376CGLU17311.28039.76528.5311.0010.86APEPATOM1377OGLU17311.62238.79029.1991.0010.81APEPATOM1378NVAL17411.40439.83027.2091.0010.16APEPATOM1379CAVAL17411.96838.74126.4161.009.96APEPATOM1380CBVAL17410.85637.81125.8461.009.93APEPATOM1381CG1VAL17410.09938.51924.7401.007.96APEPATOM1382CG2VAL17411.46036.50825.3231.007.52APEPATOM1383CVAL17412.79039.31625.2581.0011.17APEPATOM1384OVAL17412.48540.38324.7281.0011.23APEPATOM1385NGLY17513.84538.60524.8861.0010.40APEPATOM1386CAGLY17514.69239.04523.7971.008.75APEPATOM1387CGLY17515.33737.81323.2111.009.59APEPATOM1388OGLY17515.88236.99123.9491.006.65APEPATOM1389NCYS17615.29137.68521.8851.008.02APEPATOM1390CACYS17615.85336.51121.2261.007.90APEPATOM1391CCYS17616.94036.77120.1861.007.85APEPATOM1392OCYS17617.11437.89319.6931.006.33APEPATOM1393CBCYS17614.72135.70120.5821.006.21APEPATOM1394SGCYS17613.24935.55321.6411.009.41APEPATOM1395NGLY17717.67235.70319.8801.008.22APEPATOM1396CAGLY17718.73735.74418.8951.0010.14APEPATOM1397CGLY17718.61834.46618.0851.0010.70APEPATOM1398OGLY17718.18233.44618.6231.008.40APEPATOM1399NSER17818.98334.50916.8061.009.75APEPATOM1400CASER17818.88133.32515.9591.009.62APEPATOM1401CBSER17817.52333.30515.2471.0012.13APEPATOM1402OGSER17817.61433.90213.9641.0015.65APEPATOM1403CSER17819.99933.22714.9211.008.56APEPATOM1404OSER17820.59734.23114.5321.005.09APEPATOM1405NILE17920.27032.00114.4821.009.37APEPATOM1406CAILE17921.31031.74413.4981.009.01APEPATOM1407CBILE17922.67331.53114.1811.008.43APEPATOM1408CG2ILE17922.62530.29615.0541.006.63APEPATOM1409CG1ILE17923.77431.41513.1221.007.53APEPATOM1410CD1ILE17925.09332.03513.5351.008.62APEPATOM1411CILE17920.98030.51712.6501.0010.87APEPATOM1412OILE17920.50629.49713.1581.0010.47APEPATOM1413NLYS18021.21630.63211.3471.0010.60APEPATOM1414CALYS18020.95229.53610.4301.0011.05APEPATOM1415CBLYS18020.07730.0189.2691.0011.05APEPATOM1416CGLYS18018.74530.5969.7241.0013.07APEPATOM1417CDLYS18017.90231.0488.5431.0015.79APEPATOM1418CELYS18016.58031.6558.9961.0014.81APEPATOM1419NZLYS18015.62031.8027.8621.0016.66APEPATOM1420CLYS18022.29129.0299.9201.0010.53APEPATOM1421OLYS18023.13729.8179.5051.0012.20APEPATOM1422NTYR18122.49027.7169.9551.009.11APEPATOM1423CATYR18123.75627.1669.5101.008.14APEPATOM1424CBTYR18124.78627.30010.6331.006.13APEPATOM1425CGTYR18124.46026.48311.8631.008.18APEPATOM1426CD1TYR18124.98425.20112.0271.009.63APEPATOM1427CE1TYR18124.70624.45013.1721.008.66APEPATOM1428CD2TYR18123.64326.99912.8761.008.34APEPATOM1429CE2TYR18123.36026.25714.0201.007.46APEPATOM1430CZTYR18123.89624.98614.1611.008.38APEPATOM1431OHTYR18123.63424.24415.2931.008.05APEPATOM1432CTYR18123.69525.7199.0221.007.08APEPATOM1433OTYR18122.72825.0019.2621.007.97APEPATOM1434NILE18224.74325.3038.3231.008.25APEPATOM1435CAILE18224.81423.9487.8041.007.94APEPATOM1436CBILE18225.01123.9596.2931.009.43APEPATOM1437CG2ILE18224.64122.5995.7131.009.49APEPATOM1438CG1ILE18224.14725.0655.6801.009.61APEPATOM1439CD1ILE18224.50225.4294.2781.007.05APEPATOM1440CILE18225.96123.1848.4451.008.67APEPATOM1441OILE18227.11223.5888.3331.008.62APEPATOM1442NGLN18325.64222.0849.1221.006.39APEPATOM1443CAGLN18326.65721.2719.7761.007.60APEPATOM1444CBGLN18326.48521.31311.3041.008.26APEPATOM1445CGGLN18327.18420.16012.0591.0011.17APEPATOM1446CDGLN18326.84220.10513.5601.0010.23APEPATOM1447OE1GLN18325.92720.77914.0291.0013.96APEPATOM1448NE2GLN18327.57819.29314.3041.009.47APEPATOM1449CGLN18326.60319.8249.3081.006.95APEPATOM1450OGLN18325.65319.0969.6021.004.41APEPATOM1451NGLU18427.62419.4048.5761.006.92APEPATOM1452CAGLU18427.68518.0258.1231.007.66APEPATOM1453CBGLU18427.88517.1209.3491.009.14APEPATOM1454CGGLU18429.11017.55110.1721.008.94APEPATOM1455CDGLU18429.20716.91211.5581.0012.48APEPATOM1456OE1GLU18428.23516.96312.3401.0013.16APEPATOM1457OE2GLU18430.27816.36111.8691.0015.28APEPATOM1458CGLU18426.44717.6457.3161.006.36APEPATOM1459OGLU18425.85816.5817.4931.004.35APEPATOM1460NLYS18526.08918.5606.4171.008.43APEPATOM1461CALYS18524.95618.4515.5041.0010.57APEPATOM1462CBLYS18525.05417.1654.6851.0012.55APEPATOM1463CGLYS18525.70517.3713.3311.0016.19APEPATOM1464CDLYS18526.93016.4983.1731.0020.18APEPATOM1465CELYS18526.78315.5531.9901.0022.27APEPATOM1466NZLYS18525.36015.1821.7441.0025.09APEPATOM1467CLYS18523.57118.5676.1311.0010.92APEPATOM1468OLYS18522.56618.2195.5091.0010.67APEPATOM1469NTRP18623.51919.0627.3621.0010.21APEPATOM1470CATRP18622.25019.2528.0391.009.13APEPATOM1471CBTRP18622.29718.6779.4611.008.92APEPATOM1472CGTRP18622.10517.1739.5631.007.23APEPATOM1473CD2TRP18620.88316.4339.3801.007.44APEPATOM1474CE2TRP18621.17915.0709.6241.007.10APEPATOM1475CE3TRP18619.56916.7879.0361.007.24APEPATOM1476CD1TRP18623.05716.2539.8951.007.55APEPATOM1477NE1TRP18622.51014.9919.9341.007.21APEPATOM1478CZ2TRP18620.20914.0619.5361.004.65APEPATOM1479CZ3TRP18618.60215.7768.9501.005.07APEPATOM1480CH2TRP18618.93414.4309.1981.004.34APEPATOM1481CTRP18622.02920.7578.0971.009.22APEPATOM1482OTRP18622.89521.4958.5471.0010.78APEPATOM1483NHIS18720.87621.2147.6221.0011.53APEPATOM1484CAHIS18720.55522.6377.6421.0010.59APEPATOM1485CBHIS18719.77323.0056.3751.0011.28APEPATOM1486CGHIS18720.38122.4555.1191.0012.80APEPATOM1487CD2HIS18720.51121.1834.6741.0013.63APEPATOM1488ND1HIS18720.98423.2544.1701.0014.63APEPATOM1489CE1HIS18721.46322.4973.1981.0014.97APEPATOM1490NE2HIS18721.18921.2363.4801.0014.98APEPATOM1491CHIS18719.73822.9108.9041.008.99APEPATOM1492OHIS18718.63622.4089.0581.0010.40APEPATOM1493NLYS18820.28723.7009.8171.0010.12APEPATOM1494CALYS18819.59323.97011.0681.009.30APEPATOM1495CBLYS18820.40323.41312.2511.0010.66APEPATOM1496CGLYS18821.39522.31411.9091.007.22APEPATOM1497CDLYS18821.62721.42213.1181.008.34APEPATOM1498CELYS18822.69620.36912.8711.009.03APEPATOM1499NZLYS18823.26819.86514.1621.0013.31APEPATOM1500CLYS18819.28925.42811.3491.007.81APEPATOM1501OLYS18819.98826.32810.8901.009.61APEPATOM1502NHIS18918.21625.64612.0971.009.34APEPATOM1503CAHIS18917.82326.97712.5321.009.56APEPATOM1504CBHIS18916.39127.31512.1191.009.45APEPATOM1505CGHIS18916.03328.75612.3311.0011.25APEPATOM1506CD2HIS18916.73929.77712.8701.0011.06APEPATOM1507ND1HIS18914.82229.29111.9461.0013.10APEPATOM1508CE1HIS18914.80030.57912.2371.0011.47APEPATOM1509NE2HIS18915.95030.90012.7991.0012.33APEPATOM1510CHIS18917.91126.90414.0491.009.30APEPATOM1511OHIS18917.26526.06014.6711.009.01APEPATOM1512NTYR19018.71727.78114.6351.0010.00APEPATOM1513CATYR19018.92827.81616.0801.009.41APEPATOM1514CBTYR19020.43327.74516.3431.0011.08APEPATOM1515CGTYR19020.87227.61817.7881.0011.54APEPATOM1516CD1TYR19020.01727.12418.7771.0011.05APEPATOM1517CE1TYR19020.45826.98320.1081.0010.64APEPATOM1518CD2TYR19022.17327.97118.1571.0011.80APEPATOM1519CE2TYR19022.61827.83519.4671.0012.63APEPATOM1520CZTYR19021.76727.34220.4351.0011.58APEPATOM1521OHTYR19022.25727.19021.7131.0012.58APEPATOM1522CTYR19018.33729.07616.7161.008.19APEPATOM1523OTYR19018.80330.18416.4561.006.34APEPATOM1524NLEU19117.31328.89517.5491.007.47APEPATOM1525CALEU19116.65630.01118.2261.008.92APEPATOM1526CBLEU19115.15130.00117.9261.008.75APEPATOM1527CGLEU19114.30831.10118.5991.0011.51APEPATOM1528CD1LEU19114.54032.44417.9161.009.71APEPATOM1529CD2LEU19112.83130.72418.5411.009.30APEPATOM1530CLEU19116.89029.99619.7431.009.50APEPATOM1531OLEU19116.66728.98820.4161.0011.01APEPATOM1532NVAL19217.34731.12820.2661.009.30APEPATOM1533CAVAL19217.62931.29121.6901.0010.13APEPATOM1534CBVAL19219.14531.53621.9231.0010.96APEPATOM1535CG1VAL19219.38732.04423.3441.0011.81APEPATOM1536CG2VAL19219.93430.26721.6591.0011.43APEPATOM1537CVAL19216.87532.51022.2311.0010.34APEPATOM1538OVAL19217.07833.62121.7451.0010.10APEPATOM1539NCYS19316.00932.31523.2261.008.18APEPATOM1540CACYS19315.27633.44223.8101.008.37APEPATOM1541CCYS19315.60033.60925.2961.008.44APEPATOM1542OCYS19315.51432.65026.0611.006.27APEPATOM1543CBCYS19313.76233.25823.6491.009.08APEPATOM1544SGCYS19313.06233.55621.9851.008.61APEPATOM1545NASN19415.97834.82625.6941.008.32APEPATOM1546CAASN19416.31035.13327.0861.007.48APEPATOM1547CBASN19417.58235.99127.1491.008.69APEPATOM1548CGASN19418.84035.19026.8681.006.67APEPATOM1549OD1ASN19418.77233.99526.5961.009.58APEPATOM1550ND2ASN19419.98935.84326.9321.004.71APEPATOM1551CASN19415.14035.85927.7661.007.75APEPATOM1552OASN19414.54036.76027.1751.005.25APEPATOM1553NTYR19514.83435.47529.0091.007.83APEPATOM1554CATYR19513.69936.04729.7491.007.00APEPATOM1555CBTYR19512.73634.92430.1381.006.30APEPATOM1556CGTYR19512.18034.18028.9491.008.42APEPATOM1557CD1TYR19512.91833.17528.3291.007.89APEPATOM1558CE1TYR19512.43632.51027.2191.008.54APEPATOM1559CD2TYR19510.93434.50128.4221.006.50APEPATOM1560CE2TYR19510.43833.83527.3001.008.99APEPATOM1561CZTYR19511.19932.83726.7071.008.08APEPATOM1562OHTYR19510.72432.14225.6171.008.91APEPATOM1563CTYR19514.04736.86030.9991.006.48APEPATOM1564OTYR19514.84036.42231.8221.007.26APEPATOM1565NGLY19613.41838.02531.1541.006.42APEPATOM1566CAGLY19613.70938.86732.3031.006.88APEPATOM1567CGLY19612.55839.43133.1311.008.03APEPATOM1568OGLY19611.64940.07532.5961.008.29APEPATOM1569NPRO19712.54139.15734.4451.006.50APEPATOM1570CDPRO19711.52539.69835.3631.007.41APEPATOM1571CAPRO19713.53638.34335.1531.006.68APEPATOM1572CBPRO19713.30038.68836.6101.006.91APEPATOM1573CGPRO19711.85639.04136.6721.006.39APEPATOM1574CPRO19713.26636.86834.8541.006.69APEPATOM1575OPRO19712.25536.53734.2381.006.02APEPATOM1576NSER19814.15335.97535.2861.007.01APEPATOM1577CASER19813.95334.55735.0011.007.41APEPATOM1578CBSER19815.23333.75535.3031.004.71APEPATOM1579OGSER19815.55833.75236.6821.0011.52APEPATOM1580CSER19812.76533.92735.7171.007.39APEPATOM1581OSER19812.16134.52836.6051.006.19APEPATOM1582NGLY19912.42332.71635.2891.007.86APEPATOM1583CAGLY19911.33231.97735.8931.008.49APEPATOM1584CGLY19911.89430.62236.2741.008.12APEPATOM1585OGLY19913.11030.45036.3061.008.44APEPATOM1586NASN20011.02229.67036.5701.009.40APEPATOM1587CAASN20011.43328.31736.9291.0011.14APEPATOM1588CBASN20012.34427.74235.8441.0011.38APEPATOM1589CGASN20011.58127.36534.5911.0012.36APEPATOM1590OD1ASN20010.36027.47834.5501.0013.35APEPATOM1591ND2ASN20012.29326.91933.5661.0010.02APEPATOM1592CASN20012.10228.17738.2961.0012.89APEPATOM1593OASN20013.01927.37338.4771.0012.94APEPATOM1594NPHE20111.63228.95739.2621.0014.46APEPATOM1595CAPHE20112.15728.89040.6221.0016.00APEPATOM1596CBPHE20111.94730.22441.3391.0014.75APEPATOM1597CGPHE20112.80531.33840.8111.0013.67APEPATOM1598CD1PHE20112.26732.31139.9821.0013.83APEPATOM1599CD2PHE20114.15131.42141.1571.0017.70APEPATOM1600CE1PHE20113.04733.35039.5051.0014.74APEPATOM1601CE2PHE20114.94832.46040.6851.0017.45APEPATOM1602CZPHE20114.39433.42739.8571.0017.09APEPATOM1603CPHE20111.34727.78641.3041.0016.58APEPATOM1604OPHE20110.12427.87641.3851.0016.44APEPATOM1605NLYS20212.02626.75541.7971.0018.81APEPATOM1606CALYS20211.35125.61742.4211.0021.27APEPATOM1607CBLYS20212.38624.58842.8831.0025.00APEPATOM1608CGLYS20212.31423.27442.1011.0029.78APEPATOM1609CDLYS20213.21522.19742.6981.0031.92APEPATOM1610CELYS20212.57420.81642.6091.0032.51APEPATOM1611NZLYS20212.13820.30443.9441.0030.68APEPATOM1612CLYS20210.36525.89943.5551.0021.29APEPATOM1613OLYS2029.34725.21843.6761.0022.50APEPATOM1614NASN20310.64226.89444.3851.0020.90APEPATOM1615CAASN2039.72627.19045.4851.0022.00APEPATOM1616CBASN20310.52027.65746.7111.0023.02APEPATOM1617CGASN20311.27428.95346.4661.0023.60APEPATOM1618OD1ASN20311.55929.69847.4011.0024.95APEPATOM1619ND2ASN20311.60529.22345.2091.0025.38APEPATOM1620CASN2038.65628.23245.1341.0020.82APEPATOM1621OASN2038.09628.87746.0251.0020.77APEPATOM1622NGLU2048.36328.38443.8451.0016.60APEPATOM1623CAGLU2047.38229.37243.4141.0017.29APEPATOM1624CBGLU2048.09330.55042.7371.0017.84APEPATOM1625CGGLU2049.30331.08443.4891.0017.21APEPATOM1626CDGLU2049.80132.40842.9371.0017.35APEPATOM1627OE1GLU2049.15732.96342.0231.0015.88APEPATOM1628OE2GLU20410.84232.89543.4221.0017.79APEPATOM1629CGLU2046.29828.84242.4751.0017.89APEPATOM1630OGLU2046.38327.72741.9631.0016.49APEPATOM1631NGLU2055.28329.67242.2511.0019.78APEPATOM1632CAGLU2054.15929.33541.3831.0020.21APEPATOM1633CBGLU2052.85129.85441.9921.0023.42APEPATOM1634CGGLU2052.60931.34741.7721.0028.77APEPATOM1635CDGLU2052.87432.17643.0221.0032.85APEPATOM1636OE1GLU2053.94231.98443.6601.0031.69APEPATOM1637OE2GLU2052.00833.01943.3641.0032.48APEPATOM1638CGLU2054.34829.95640.0091.0018.17APEPATOM1639OGLU2054.91831.03939.8911.0015.97APEPATOM1640NLEU2063.86329.27538.9741.0016.44APEPATOM1641CALEU2063.98329.78537.6171.0015.66APEPATOM1642CBLEU2063.27528.86036.6261.0015.15APEPATOM1643CGLEU2063.86927.47836.3681.0012.15APEPATOM1644CD1LEU2063.18926.87135.1731.009.94APEPATOM1645CD2LEU2065.37027.57936.1481.0010.37APEPATOM1646CLEU2063.33331.15537.5611.0015.68APEPATOM1647OLEU2063.92832.12537.0761.0016.18APEPATOM1648NTYR2072.10531.21738.0651.0014.52APEPATOM1649CATYR2071.33232.45138.0901.0015.05APEPATOM1650CBTYR2070.74232.74636.7051.0012.49APEPATOM1651CGTYR207−0.04631.60436.0831.0012.80APEPATOM1652CD1TYR207−1.37931.36536.4411.0013.16APEPATOM1653CE1TYR207−2.11330.32735.8561.0012.28APEPATOM1654CD2TYR2070.53330.77435.1201.0014.06APEPATOM1655CE2TYR207−0.19529.73134.5281.0014.10APEPATOM1656CZTYR207−1.51329.51534.9031.0012.49APEPATOM1657OHTYR207−2.22028.48734.3321.0012.47APEPATOM1658CTYR2070.20632.33139.1131.0014.67APEPATOM1659OTYR207−0.08831.24439.5951.0015.57APEPATOM1660NGLN208−0.42533.45339.4321.0015.61APEPATOM1661CAGLN208−1.52333.46640.3931.0015.81APEPATOM1662CBGLN208−1.73334.89240.8961.0014.83APEPATOM1663CGGLN208−2.44034.99442.2311.0016.28APEPATOM1664CDGLN208−2.84336.41842.5641.0016.96APEPATOM1665OE1GLN208−2.07437.36442.3521.0016.63APEPATOM1666NE2GLN208−4.05136.58143.0861.0016.21APEPATOM1667CGLN208−2.80932.94739.7391.0017.01APEPATOM1668OGLN208−3.24333.46938.7171.0014.76APEPATOM1669NTHR209−3.42231.92140.3161.0019.39APEPATOM1670CATHR209−4.64931.39239.7361.0023.16APEPATOM1671CBTHR209−4.79229.87739.9531.0023.21APEPATOM1672OG1THR209−5.01029.61741.3431.0027.25APEPATOM1673CG2THR209−3.55929.15139.4961.0023.39APEPATOM1674CTHR209−5.87332.06340.3401.0025.33APEPATOM1675OTHR209−5.86832.45541.5051.0025.28APEPATOM1676NLYS210−6.92232.18839.5361.0027.53APEPATOM1677CALYS210−8.16032.80139.9861.0029.31APEPATOM1678CBLYS210−8.49134.01939.1221.0030.03APEPATOM1679CGLYS210−8.64333.69637.6471.0028.82APEPATOM1680CDLYS210−9.57534.67536.9631.0029.76APEPATOM1681CELYS210−8.89735.34535.7711.0028.88APEPATOM1682NZLYS210−9.50036.66935.4371.0028.04APEPATOM1683CLYS210−9.27231.77539.8731.0031.66APEPATOM1684OT1LYS210−10.17131.77540.7441.0033.57APEPATOM1685OT2LYS210−9.22430.98138.9061.0033.91APEPATOM1686OH2WAT100128.32131.88430.0231.004.99AWATATOM1687OH2WAT10020.07028.63738.2801.005.19AWATATOM1688OH2WAT10039.57434.98440.1991.006.03AWATATOM1689OH2WAT100413.42328.2414.6741.006.60AWATATOM1690OH2WAT100525.59314.2118.9051.009.08AWATATOM1691OH2WAT1006−5.94828.13330.3781.007.55AWATATOM1692OH2WAT100713.72927.74630.5991.008.15AWATATOM1693OH2WAT100822.45333.97426.3651.006.87AWATATOM1694OH2WAT100911.64446.10727.5941.004.61AWATATOM1695OH2WAT1010−0.65026.16233.9011.008.02AWATATOM1696OH2WAT10118.75523.06034.4551.0010.12AWATATOM1697OH2WAT101210.78939.3488.2881.003.59AWATATOM1698OH2WAT101328.09115.73714.9121.008.00AWATATOM1699OH2WAT101516.39743.67819.3871.002.04AWATATOM1700OH2WAT101614.31129.73132.1271.005.53AWATATOM1701OH2WAT10172.57041.16721.5451.005.42AWATATOM1702OH2WAT101825.36428.50621.3321.009.41AWATATOM1703OH2WAT101926.10750.46126.2141.005.64AWATATOM1704OH2WAT102030.46946.59834.2071.007.23AWATATOM1705OH2WAT102130.25120.9698.9041.0013.93AWATATOM1706OH2WAT1022−4.47637.48634.0431.007.76AWATATOM1707OH2WAT102331.77027.79419.0201.0013.29AWATATOM1708OH2WAT102417.64444.09130.2281.008.53AWATATOM1709OH2WAT1025−6.20719.85235.2531.0017.83AWATATOM1710OH2WAT102614.73724.65710.9541.0012.71AWATATOM1711OH2WAT10273.82443.79024.6741.0011.15AWATATOM1712OH2WAT10287.49917.20926.8601.0018.25AWATATOM1713OH2WAT10290.96825.19921.2211.0013.34AWATATOM1714OH2WAT103011.73836.46238.8071.0014.39AWATATOM1715OH2WAT10315.64834.01438.4271.009.11AWATATOM1716OH2WAT10321.66414.32037.3281.0015.77AWATATOM1717OH2WAT103331.94028.80210.7551.008.72AWATATOM1718OH2WAT10345.83222.09818.1711.006.17AWATATOM1719OH2WAT103533.70130.50931.9741.0018.85AWATATOM1720OH2WAT103629.16534.66837.4181.0010.68AWATATOM1721OH2WAT1037−0.40743.48929.4181.008.15AWATATOM1722OH2WAT103830.86144.58926.3201.0013.36AWATATOM1723OH2WAT10398.34541.08137.7781.0013.31AWATATOM1724OH2WAT104010.89522.81523.3991.0020.54AWATATOM1725OH2WAT104131.50342.50127.9421.0012.75AWATATOM1726OH2WAT1042−4.12317.55626.9271.006.26AWATATOM1727OH2WAT104323.63125.35017.6181.0016.68AWATATOM1728OH2WAT1044−9.26319.78928.7691.0017.07AWATATOM1729OH2WAT10452.68126.18840.0941.0010.27AWATATOM1730OH2WAT10466.15733.28140.8761.0010.99AWATATOM1731OH2WAT10471.41142.30511.3571.0012.65AWATATOM1732OH2WAT104811.02743.1288.8361.0013.35AWATATOM1733OH2WAT10498.16326.6379.3711.009.12AWATATOM1734OH2WAT105030.81252.89721.3671.005.26AWATATOM1735OH2WAT1051−1.05638.90621.5941.0021.26AWATATOM1736OH2WAT105223.48437.80638.5231.005.01AWATATOM1737OH2WAT105316.09123.2199.1321.009.59AWATATOM1738OH2WAT105410.51544.72416.2021.0021.22AWATATOM1739OH2WAT10553.85842.45719.1881.0018.71AWATATOM1740OH2WAT105620.76738.30129.0921.007.32AWATATOM1741OH2WAT105731.45037.71733.7511.0012.78AWATATOM1742OH2WAT1058−6.46915.55629.8851.0018.83AWATATOM1743OH2WAT105919.56932.50035.5671.0013.66AWATATOM1744OH2WAT106012.88332.20345.0181.0019.55AWATATOM1745OH2WAT106116.66638.81139.2301.0012.36AWATATOM1746OH2WAT10621.62724.66138.5971.0011.62AWATATOM1747OH2WAT1063−3.79723.48020.4621.0013.70AWATATOM1748OH2WAT106419.66243.90920.5831.0017.87AWATATOM1749OH2WAT106528.95936.78818.9811.0020.15AWATATOM1750OH2WAT106615.03447.18624.9091.007.01AWATATOM1751OH2WAT10671.47945.14016.4621.0015.19AWATATOM1752OH2WAT1068−9.15926.37432.7281.0010.66AWATATOM1753OH2WAT106918.34340.02615.7191.0011.74AWATATOM1754OH2WAT1070−4.92634.88322.7651.005.57AWATATOM1755OH2WAT107111.43944.25034.4451.0017.87AWATATOM1756OH2WAT107222.34633.15738.5151.0015.02AWATATOM1757OH2WAT107316.43121.026−0.7351.0017.18AWATATOM1758OH2WAT107417.27313.0972.7691.0016.44AWATATOM1759OH2WAT107520.71741.15818.8751.0011.16AWATATOM1760OH2WAT107613.42919.16510.1211.009.99AWATATOM1761OH2WAT107722.25323.11028.0971.0014.11AWATATOM1762OH2WAT1078−1.72914.63427.8511.009.23AWATATOM1763OH2WAT107916.19636.4539.9361.0018.57AWATATOM1764OH2WAT108026.77440.94039.1761.0015.71AWATATOM1765OH2WAT108127.99620.2665.3571.003.12AWATATOM1766OH2WAT108214.34544.9039.8281.0016.53AWATATOM1767OH2WAT1083−6.95619.54924.6671.0013.41AWATATOM1768OH2WAT10846.67722.67616.3701.009.78AWATATOM1769OH2WAT108524.05517.30714.2791.009.70AWATATOM1770OH2WAT108632.34829.00020.5001.0015.00AWATATOM1771OH2WAT1087−6.42134.30642.8561.0013.87AWATATOM1772OH2WAT108828.80626.18427.5681.0016.70AWATATOM1773OH2WAT10899.35417.47543.9141.0016.99AWATATOM1774OH2WAT109030.67220.87612.9871.0021.44AWATATOM1775OH2WAT1091−7.79523.65435.1981.0012.77AWATATOM1776OH2WAT10926.67542.6637.6351.0017.23AWATATOM1777OH2WAT109314.34849.24734.2291.0010.41AWATATOM1778OH2WAT1094−2.48140.06524.8021.0016.54AWATATOM1779OH2WAT1095−5.18439.22918.8381.0028.58AWATATOM1780OH2WAT1096−6.28229.16517.2081.0027.91AWATATOM1781OH2WAT10973.52619.04119.7131.0016.33AWATATOM1782OH2WAT1098−5.49040.33627.6661.0020.30AWATATOM1783OH2WAT10995.79143.55430.4341.0014.00AWATATOM1784OH2WAT110010.08534.2429.3521.0019.88AWATATOM1785OH2WAT110122.75237.4878.3251.0016.96AWATATOM1786OH2WAT110222.36440.02038.1291.0015.64AWATATOM1787OH2WAT110333.66637.53719.7561.0023.15AWATATOM1788OH2WAT110436.57934.63823.2561.0016.03AWATATOM1789OH2WAT110531.64531.13611.9711.0018.60AWATATOM1790OH2WAT110614.82326.51941.6941.0015.23AWATATOM1791OH2WAT110713.63821.3179.3751.0012.16AWATATOM1792OH2WAT110833.91348.93932.6901.0011.76AWATATOM1793OH2WAT110933.41548.32634.4901.0020.71AWATATOM1794OH2WAT1110−8.56041.28743.7251.0012.63AWATATOM1795OH2WAT111122.65624.2090.8841.0017.46AWATATOM1796OH2WAT11122.71641.25215.0631.0019.18AWATATOM1797OH2WAT111330.63531.0077.7141.0010.21AWATATOM1798OH2WAT111414.81522.01039.0231.0027.49AWATATOM1799OH2WAT111533.28647.30324.0071.0012.66AWATATOM1800OH2WAT111614.04233.41210.6221.0010.91AWATATOM1801OH2WAT111720.19527.49932.6581.0018.38AWATATOM1802OH2WAT111831.21517.82513.6781.0017.20AWATATOM1803OH2WAT111930.83121.03011.0861.0017.97AWATATOM1804OH2WAT112030.91027.31125.2841.0017.48AWATATOM1805OH2WAT11216.25913.35526.0821.0021.34AWATATOM1806OH2WAT112234.78029.65915.0891.0023.24AWATATOM1807OH2WAT112333.17028.24223.4881.0016.58AWATATOM1808OH2WAT11240.91340.67241.4551.0017.75AWATATOM1809OH2WAT112525.39336.68942.2661.0022.18AWATATOM1810OH2WAT112621.92340.74815.0351.0020.08AWATATOM1811OH2WAT1127−1.33928.43321.0941.0017.33AWATATOM1812OH2WAT112822.05828.76933.3801.0022.93AWATATOM1813OH2WAT11292.23223.03517.6631.0013.73AWATATOM1814OH2WAT11304.83440.22840.1171.0039.84AWATATOM1815OH2WAT113116.18227.9371.6921.009.10AWATATOM1816OH2WAT113236.69643.32233.6621.0014.41AWATATOM1817NANAT500−4.31215.33228.3741.0011.67ANATEND

[0247] The solvent accessibilities of Ves v 5 amino acid residues are given in Table 7.

11TABLE 7Surface Exposure of Ves v 5 amino acidsNOAASolv exp3E0.8024A0.0605E0.3906F0.8687N0.4848N0.5559Y0.03310C0.41211K0.97812I0.22513K0.95114C0.03815L0.71416K1.00017G0.14318G0.27519V0.44520H0.01621T0.00022A0.04923C0.20924K0.48925Y0.28026G0.35227S0.15928L0.42329K0.79730P0.23131N0.39632C0.05533G0.42934N0.77535K0.29736V0.48937V0.28038V0.37939S0.29140Y0.59341G0.16542L0.12143T0.42344K0.97845Q0.53846E0.26447K0.39648Q0.59349D0.30250I0.00051L0.19852K0.61553E0.17054H0.00055N0.11556D0.44557F0.02758R0.00059Q0.19860K0.40761I0.00062A0.03363R0.95664G0.14865L0.59366E0.00567T0.61068R0.33569G0.11070N0.54971P0.36372G0.17073P0.44074Q0.00575P0.20976P0.23677A0.02278K0.77579N0.23680M0.06681K0.58882N0.50083L0.01684V0.46285W0.27586N0.16587D0.62188E0.24789L0.00590A0.05591Y0.11592V0.00593A0.00094Q0.15995V0.01196W0.07797A0.00098N0.00599Q0.027100C0.027101Q0.577102Y0.687103G0.110104H0.549105D0.022106T0.429107C0.000108R0.203109D0.093110V0.044111A0.500112K0.824113Y0.209114Q0.423115V0.011116G0.011117Q0.066118N0.005119V0.022120A0.016121L0.198122T0.198123G0.231124S0.236125T0.610126A0.253127A0.379128K0.857129Y0.352130D0.220131D0.495132P0.033133V0.137134K0.654135L0.000136V0.000137K0.538138M0.473139W0.016140E0.071141D0.341142E0.154143V0.000144K0.560145D0.390146Y0.044147N0.165148P0.214149K0.868150K0.604151K0.753152F0.071153S0.302154G0.192155N0.121156D0.379157F0.819158L0.714159K0.533160T0.000161G0.077162H0.231163Y0.000164T0.000165Q0.011166M0.000167V0.000168W0.005169A0.011170N0.429171T0.000172K0.451173E0.165174V0.000175G0.000176C0.016177G0.000178S0.016179I0.000180K0.214181Y0.016182I0.275183Q0.231184E0.841185K0.989186W0.665187H0.159188K0.203189H0.011190Y0.000191L0.000192V0.000193C0.000194N0.000195Y0.000196G0.000197P0.225198S0.110199G0.027200N0.308201F0.341202K0.824203N0.797204E0.374205E0.511206L0.055207Y0.082208Q0.566209T0.473210K0.962

EXAMPLE 10

Alignment of Ag 5s

[0248] An alignment of selected antigen 5 sequences from Vespula, Dolichovespula, stes and Solenopsis (fire ants) is shown in FIG. 12. Vespula, Dolichovespula, Polistes all belong to the Vespidae family. The figure also includes the secondary structural elements of Ves v 5. When considering only the Vespula antigen 5s a very high degree of surface conservation is observed (FIG. 5), the conservation of residues being almost evenly distributed with only a few non-conserved residues scattered over the molecule.

[0249] In contrast, the surfaces conserved, when comparing sequences from the Vespula and Polistes genera, are restricted to 5 regions with solvent accessible areas of 392 Å2, 585 Å2, 589 Å2, 673 Å2 and 1053 Å2, respectively. Solvent accessibility was calculated using the NACCESS program (S. J. Hubbard and J. M. Thornton, 1992, NACCESS. (v2.1.1) Department of Biochemistry and Molecular Biology, University College London) with a probe radius of 1.4 Å. Similarly, five surface patches corresponding to the 5 surface patches conserved between Vespula and Polistes, were conserved between Vespula and Vespa/Dolichovespula. In the latter case the areas are 280 Å2, 496 Å2, 730 Å2, 803 Å2 and 1043 Å2, respectively. The residues contributing to one surface patch are primarily from the beginning of the B strand and from helix IV, the residues contributing to a second surface patch are primarily from the A strand and the loop between helix II and strand B, the residues contributing to a third surface patch is primarily from helix I and its surroundings and from the end of helix II, the residues contributing to a fourth surface patch is mainly of N-terminal origin while a fifth surface patch is dominated by residues from the end of helix I and the loop between helix I and the A strand.

DISCUSSION

[0250] Crystallographic studies of protein antigen-antibody complexes have shown that the contact residues of an epitope may contain as many as 17 residues on the surface of an antigen, and that these residues may, or may not, be contiguous to each other in the peptide chain (Davies et al., 1996, Proc. Natl Acad. Sci USA, 93:7). Epitope mapping of lysozyme with monoclonal antibodies have shown that the entire surface of a protein is potentially antigenic (Newmann et al., 1992, J. Immunol. 149:3260). Thus the hybrids with {fraction (1/10)} to ¾ of yellow jacket antigen 5, will have fewer epitopes than the parent molecule.

[0251] The CD spectral data in FIG. 7 suggest that the hybrids have secondary structures closely similar, if not identical, with those of vespid antigen 5s. The inhibition data in FIGS. 8 and 9 with Ves v 5-specific human and mouse antibodies and the antibody binding data in Table 3 with hybrid-specific antibodies suggest that the hybrids have tertiary structures closely similar or identical with that of Ves v 5, as these antibodies do not bind the denatured Ves v 5. Additional evidence came from screening with 17 monoclonal mouse IgG1 antibodies specific for the natural Ves v 5, six of which bound the N-terminal hybrid PV1-46. Therefore these data indicate that the hybrids contain the discontinuous B cell epitopes of Ves v 5.

[0252] The inhibition data with polyclonal antibodies and the binding data with monoclonal antibodies indicate that the dominant B cell epitopes of Ves v 5 are in its N-terminal region. Inspection of the structure of Ves v 5 in shows that nearly all residues in the N-terminal hybrid PV1-46 are surface accessible. (See Table 7) This is in contrast to the C-terminal hybrid PV156-204, in which only segments of Ves v 5 are surface accessible. (See Table 7) This difference in surface accessibility may explain the immunodominance of the N-terminal region of antigen 5. Others have shown that the entire surface of a protein is potentially antigenic but the regions with high surface accessibility and surface protrusion are dominant (Newmann et al., 1992, J. Immunol 149:3260 and Novotny et al., 1996, Adv Prot Chem 49:149).

[0253] At present the only known way to map discontinuous epitopes is by X-ray crystallography of Ag-Ab complexes (Davies et al., 1996, Proc. Natl Acad. Sci USA, 93:7) and this requires having specific monoclonal antibodies. The discontinuous epitopes of CD39 was mapped with a series of mouse-human hybrids, mouse and human CD39 molecules have 75% sequence identity and they share limited antigenic cross-reactivity (Maliszewski et al., 1994, J. Immunol 153:3574). These findings with CD39 and antigen 5 indicate that hybrids of two homologous proteins represent a useful approach to mapping their discontinuous B cell epitopes.

[0254] Our results with hybrid Ag 5s demonstrate that hybrid allergens can have a hundred to a thousand-fold reduction in allergenicity yet retain the immunogenicity of the natural allergens. This reduction in allergenicity of hybrids is believed to be mainly due to a decrease of B cell epitope density. Each hybrid of the Examples has only a portion of the B and T cell epitopes of Ves v 5. In principle, however, a mixture of hybrids can reconstitute the complete epitope library of Ves v 5. Thus, all epitopes can be reconstituted to prepare modified allergens for use as vaccines. Our results suggest that a PV hybrid with 20-30 residues of Ves v 5 will have maximal reduction in allergenicity yet retaining immunogenicity for Ves v 5.

[0255] Many allergens have sequence homology with proteins from diverse sources (Larsen et al., 1996, J Allergy Clin Immunol 97:577). For example, vespid Ag 5s have varying degrees of sequence homology with a variety of extracellular proteins from different organisms, ranging from fungi to humans (see FIG. 12). It is known that homologous proteins of 30% sequence identity may have the same or closely similar structures (Chothia et al., 1990, Annual Review Biochem 59:1007 and Russell et al., 1994, J. Mol. Biol. 244:332). Thus, hybrids may be prepared with a variety of homologous host proteins to function as scaffolds for the guest allergen fragment of interest.

[0256] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0257] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.

12TABLE 8AllergensSIZEREFERENCE/ORGANISMALLERGENPROTEIN(kD)C/PaACCESSION NOWeed pollensAsteralesAmbrosia artemisiifoliaAmb a 1antigen E38C8, 20short ragweedAmb a 2antigen K38C8, 21Amb a 3Ra311C22Amb a 5Ra5 5C11, 23Amb a 6Ra610P24, 25Amb a 7Ra712C26Amb a ?11C27Ambrosia trifidaAmb t 5Ra5G 4.4C9, 10, 28giant ragweedArtemisia vulgarisArt v 127-29C28AmugwortArt v 235P29Art v 312P53Art v 414CHelianthus annuusHel a 13429AsunflowerHel a 2profilin 15.7CY15210Mercurialis annuaMer a 1profilin14-15CY13271CaryophyllalesSalsola kaliSal k 14343P29BRussian thistleGrass pollensPoalesCynodon dactylonBermuda grassCyn d 132C30, S83343Cyn d 7C31, X91256Cyn d 12profilin14C31a, Y08390Dactylis glomerataDac g 1AgDg132P32orchard grassDac g 211C33, S45354Dac g 3C33A, U25343Dac g 531P34Holcus lanatusHol l 1CZ27084velvet grassLolium perenneLol p 1group I27C35, 36rye grassLol p 2group II11P37, 37A, X73363Lol p 3group III11P38Lol p 5Lol p IX, Lol p Ib31/35C34, 39Lol p 11hom: trypsin inhibitor1639APhalaris aquaticaPha a 1C40, S80654canary grassPhleum pratensePhl p 127CX78813timothyPhl p 2C41, X75925Phl p 4P41APhl p 5Ag2532C42Phl p 6C43, Z27082Phl p 12profilinC44, X77583Phl p 13polygalacturonase55-60CAJ238848Poa pratensisPoa p 1group I33P46Kentucky blue grassPoa p 531/34C34, 47Sorghum halepenseSor h 1C48Johnson grassTree pollensFagalesAlnus glutinosaAln g 117C550892alderBetula verrucosaBet v 117C49,50, Z80098birchBet v 2profilin15CM65179Bet v 3CX79267Bet v 4 8CX87153, 554819Bet v 6h: isoflavone reductase 33.5CAF135 127Bet v 7cyclophilin18PP81531Carpinus betulusCar b 117C51, X66932,hornbeamX66918Castanea sativaCas s 122P52chestnutCas s 5chitinaseCas s 8lipid transfer protein 9.7p53Corylus avellanaCor a 117C54A, X70999hazelCor a 2profilin14CAF327622Quercus albaQue a 117P54White oakLamialesOleaceaeFraxinus excelsiorFra e 120P58AashLigustrum vulgareLig v 120P58AprivetOlea europeaOle e 116C59, 60oliveOle e 2profilin15-18C60AOle e 3 9.260BOle e 432PP80741Ole e 5superoxide dismutase16PP80740Ole e 610C60C, U86342Ole e 7P60D, P81430Syringa vulgarisSyr v 120P58AlilacPlantaginaceaePlantago lanceolataPla l 118PP842242English plantainPinalesCryptomeria japonicaCry j 141-45C55, 56sugiCry j 2C57, D29772Cupressus arizonicaCup a 143CA1243570cypressJuniperus asheiJun a 143PP81294mountain cedarJun a 2C57A, AJ404653Jun a 330P57B, P81295Juniperus oxycedrusJun o 4hom: calmodulin29C57C, AF031471prickly juniperJuniperus sabinoidesJun s50P58mountain cedarJuniperus virginianaJun v 143PP81825eastern red cedarMitesAcarus siroAca s 13fatty acid binding prot 14*CAJ006774miteBlomia tropicalisBlo t 5CU59102miteBlo t 12Bt11aCU27479Blo t 13Bt6, fatty acid bind prot.CU58106DermatophagoidesDer p 1antigen P125C61pteronyssinusDer p 214C62miteDer p 3trypsin28/30C63Der p 4amylase60P64Der p 514C65Der p 6chymotrypsin25P66Der p 722/28C67Der p 8glutathione transferaseC67ADer p 9collagenolytic serineP67Bpro.Der p 10tropomyosin36CY14906Der p 14apolipophorin like prot.CDermatophagoidesDer m 125P68microcerasmiteDermatophagoides farinaeDer f 125C69miteDer f 214C70, 71Der f 330C63Der f 10tropomyosinC72Der f 11paramyosin98C72ADer f 14mag3, apolipophorinCD17686Derf f 1598k chitinase98CAF178772Derf f 16gelsolin/villin53C71ADerf f 17Ca binding EF protein53C71AEuroglyphus mayneiEur m 14apolipophorin177 CAF149827miteLepidoglyphus destructorLep d 215C73, 74, 75storage miteLep d 5C75A, AJ250278Lep d 7C75A, AJ271058Lep d 10tropomyosinCAJ25096Lep d 13C75A, AJ250279AnimalsBos domesticusBos d 2Ag3, lipocalin20C76, L42867domestic cattleBos d 3Ca-binding S100 hom11CL39834(see also foods)Bos d 4alpha-lactalbumin 14.2CM18780Bos d 5beta-lactoglobulin 18.3CX14712Bos d 6serum albumin67CM73993Bos d 7immunoglobulin160 77Bos d 8caseins20-3077Canis familiarisCan f 125C78, 79(Canis domesticus)Can f 227C78, 79dogCan f 3albuminCS72946Equus caballusEqu c 1lipocalin25CU70823domestic horseEqu c 2lipocalin18P79A, 79BEqu c 3Ag3-X67C79C, X74045Equ c 417P79DEqu c 5AgX17PFelis domesticusFel d 1cat-138C15cat (saliva)Fel d 2albuminC79E, X84842Fel d 3cystatin11C79F, AF238996Mus musculusMus m 1MUP19C80, 81mouse (urine)Rattus norvegiusRat n 117C82, 83rat (urine)Fungi (moulds)AscomycotaDothidialesAlternaria alternataAlt a 128CU82633Alt a 225C83A, U62442Alt a 3heat shock prot. 70CU87807, U87808Alt a 4prot. disulfideisomerase57CX84217Alt a 6acid ribosomal prot. P211CX78222, U87806Alt a 7YCP4 protein22CX78225Alt a 10aldehyde dehydrogenase53CX78227, P42041Alt a 11enolase45CU82437Alt a 12acid ribosomal prot. P111CX84216Cladosporium herbarumCla h 11383B, 83CCla h 22383B, 83CCla h 3aldehyde dehydrogenase53CX78228Cla h 4acid ribosomal prot. P211CX78223Cla h 5YCP4 protein22CX78224Cla h 6enolase46CX78226Cla h 12acid ribosomal prot. P111CX85180EurotialesAspergillus flavusAsp fl 13alkaline serine protease3484Aspergillus fumigatusAsp f 118CM83781, S39330Asp f 237CU56938Asp f 3peroxisomal protein19CU20722Asp f 430CAJ001732Asp f 5metalloprotease40CZ30424Asp f 6Mn superoxide dismut. 26.5CU53561Asp f 712CAJ223315Asp f 8ribosomal prot. P211CAJ224333Asp f 934CAJ223327Asp f 10aspartic protease34CX85092Asp f 11peptidyl-prolyl isomeras2484AAsp f 12heat shock prot. P9090C85Asp f 13alkaline serine protease3484BAsp f 1516CAJ002026Asp f 1643Cg3643813Asp f 17CAJ224865Asp f 18vacuolar serine protease3484CAspergillus nigerAsp n 14beta-xylosidase105 CAF108944Asp n 18vacuolar serine protease34C84BAsp n ?85CZ84377Aspergillus oryzaeAsp o 13alkaline serine protease34CX17561Asp o 21TAKA-amylase A53CD00434, M33218PenicilliumPen b 13alkaline serine protease3386AbrevicompactumPenicillium citrinumPen c 3peroxisomal mem. prot.1886BPen c 13alkaline serine protease3386APen c 19heat shock prot. P7070CU64207Pen c 22wenolase46CAF254643Penicillium notatumPen n 13alkaline serine protease3489Pen n 18vacuolar serine protease3289Pen n 20N-acetyl glucosaminidas6887Penicillium oxalicumPen o 18vacuolar serine protease3489OnygenalesTrichophyton rubrumTri r 2C90Tri r 4serine proteaseC90Trichophyton tonsuransTri t 130P91Tri t 4serine protease83C90SaccharomycetalesCandida albicansCand a 140C88Candida boidiniiCand b 220CJ04984, J04985BasidiomycotaBasidiolelastomycetesMalassezia furfurMala f 191AMala f 2MF1, peroxisomal21CAB011804membrane proteinMala f 3MF2, peroxisomal20CAB011805membrane proteinMala f 435CMala f 5 18*CAJ011955Mala f 6 17*CAJ011956BasidiomycetesPsilocybe cubensisPsi c 1Psi c 2cyclophilin1691BCoprinus comatusCop c 1leucine zipper protein11CAJ132235shaggy capCop c 2AJ242791Cop c 3AJ242792Cop c 5AJ242793Cop c 7AJ242794InsectsAedes aegyptiiAed a 1apyrase68CL12389mosquitoAed a 237CM33157Apis melliferaApi m 1phospholipase A216C92honey beeApi m 2hyaluronidase44C93Api m 4melittin3C94Api m 67-8PBombus pennsylvanicusBom p 1phospholipase16P95bumble beeBom p 4proteaseP95Blattella germanicaBla g 1Bd90kCGerman cockroachBla g 2aspartic protease36C96Bla g 4calycin21C97Bla g 5glutathione transferase22C98Bla g 6troponin C27C98Periplaneta americanaPer a 1Cr-PIICAmerican cockroachPer a 3Cr-PI72-78C98APer a 7tropomyosin37CY14854Chironomus thummiChi t 1-9hemoglobin16C99thummiChi t 1.01component III16CP02229midgesChi t 1.02component IV16CP02230Chi t 2.0101component I16CP02221Chi t 2.0102component IA16CP02221Chi t 3component II-beta16CP02222Chi t 4component IIIA16CP02231Chi t 5component VI16CP02224Chi t 6.01component VIIA16CP02226Chi t 6.02component IX16CP02223Chi t 7component VIIB16CP02225Chi t 8component VIII16CP02227Chi t 9component X16CP02228Dolichovespula maculataDol m 1phospholipase A135C100white face hornetDol m 2hyaluronidase44C101Dol m 5antigen 523C102, 103Dolichovespula arenariaDol a 5antigen 523C104yellow hornetPolistes annulariesPol a 1phospholipase A135P105waspPol a 2hyaluronidase44P105Pol a 5antigen 523C104Polistes dominulusPol d 1Mediterranean paper waspPol d 4serine protease32-34CPol d 5P81656Polistes exclamansPol e 1phospholipase A134P107waspPol e 5antigen 523C104Polistes fuscatusPol f 5antigen 523C106waspPolistes metricusPol m 5antigen 523C106waspVespa craboVesp c 1phospholipase34P107European hornetVesp c 5antigen 523C106Vespa mandarinaVesp m 1giant asian hornetVesp m 5P81657Vespula flavopilosaVes f 5antigen 523C106yellowjacketVespula germanicaVes g 5antigen 523C106yellowjacketVespula maculifronsVes m 1phospholipase A1 33.5C108yellowjacketVes m 2hyaluronidase44P109Ves m 5antigen 523C104Vespula pennsylvanicaVes p 5antigen 523C106yellowjacketVespula squamosaVes s 5antigen 523C106yellowjacketVespula viduaVes vi 5antigen 523C106waspVespula vulgarisVes v 1phospholipase A135C105AyellowjacketVes v 2hyaluronidase44P105AVes v 3antigen 523C104Myrmecia pilosulaMyr p 1CX70256Australian jumper antMyr p 2CS81785Solenopsis geminataSol g 2tropical fire antSol g 4Solenopsis invictaSol i 213C110, 111fire antSol i 324C110Sol i 413C110Solenopsis saevissimaSol s 2Brazilian fire antFoodsGadus callariasGad c 1allergen M12C112, 113codSalmo salarSal s 1parvalbumin12CX97824Atlantic salmonBos domesticusBos d 4alpha-lactalbumin 14.2CM18780domestic cattleBos d 5beta-lactoglobulin 18.3CX14712(milk)Bos d 6serum albumin67CM73993Bos d 7immunoglobulin160 77Bos d 8caseins20-3077Gallus domesticusGal d 1ovomucoid28C114, 115chickenGal d 2ovalbumin44C114, 115Gal d 3Ag22, conalbumin78C114, 115Gal d 4lysozyme14C114, 115Gal d 5serum albumin69CX60688Metapenaeus ensisMet e 1tropomyosinCU08008shrimpPenaeus aztecusPen a 1trapomyosin36P116shrimpPenaeus indicusPen i 1tropomyosin34C117shrimpTodarodes pacificusTod p 1tropamyasin38P117AsquidHaliotis midaeHal m 149117BabaloneApium graveolensApi g 1hom: Bet v 1 16*CZ48967celeryApi g 4profilinAF129423Api g 555/58PP81943Brassica junceaBra j 12S albumin14C118oriental mustardBrassica rapaBra r 2hom: prohevein25P81729turnipHordeum vulgareHor v 15BMAI-115C119barleyZea maysZea m 14lipid transfer protein 9PP19656maize, cornOryza sativaOry s 1CU31771riceCorylus avellanaCor a 1.0401hom: Bet v 117CAF136945hazelnutMalus domesticaMal d 1hom: Bet v 1CX83672appleMal d 2hom: thaumatinCAJ243427Mal d 3lipid transfer protein 9CPyrus communisPyr c 1hom: Bet v 118CAF05730pearPyr c 4profilin14CAF129424Pyr c 5hom: isoflavone 33.5CAF071477reductasPersea americanaPers a 1endochitinase32CZ78202avocadoPrunus armeniacaPru ar 1hom: Bet v 1CU93165apricotPru ar 3lipid transfer protein 9PPrunus aviumPru av 1hom: Bet v 1CU66076sweet cherryPru av 2hom: thaumatinCU32440Pru av 3lipid transfere protein10CAF221501Pru av 4profilin15CAF129425Prunus domesticaPru d 3lipid transfer protein 9P119AEuropean plumPrunus persicaPru p 3lipid transfer protein10PP81402peachVitis viniferaVit v 1lipid transfer protein 9PP80274grapeMusa x paradisiacaMus xp 1profilin15CAF377948bananaAnanas comosusAna c 1profilin15CAF377949pineappleLichti chinensisLit c 1profilin15CAY049013litchiSinapis albaSin a 12S albumin14C120yellow mustardGlycine maxGly m 1HPS 7P121soybeanGly m 2 8PA57106Gly m 3profilin14CAJ223982Arachis hypogaeaAra h 1vicilin 63.5CL34402peanutAra h 2conglutin17CL77197Ara h 3glycinin60CAF093541Ara h 4glycinin37CAF086821Ara h 5profilin15CAF059616Ara h 6hom: conglutin15CAF092846Ara h 7hom: conglutin15CAF091737Actinidia chinensisAct c 1cysteine protease30PP00785kiwiCapsicum annumCap a 1wosmotin-like protein23cAJ297410bell pepperSolanum tuberosumSola t 1patatin43PP15476potatoSola t 2cathepsin D inhibitor21PP 16348Sola t 3cys. protease inhibitor21PP20347Sola t 4asp. protease inhibitor16 + 4PP30941Bertholletia excelsaBer e 12S albumin 9CP04403, M17146Brazil nutJuglans regiaJug r 12S albuminCU66866English walnutJug r 2vicilin44CAF066055Ricinus communisRic c 12S albuminCP01089Castor beanSesamum indicumSes i 12S albumin 9C121A, AF240005sesameSes i 22S albumin 7CAF091841Ses i 37S vicilin-like globulin45CAF240006Cucumis meloCuc m 1serine protease66CD32206muskmelonAdditional:Anisakis simplexAni s 124P121B, A59069nematodeAni s 2paramyosin97CAF173004Ani s 3tropomyosin41C121C, Y19221Ascaris suumAsc s 110P122wormDendronephthya nipponicaDen n 153P122Asoft coralHevea brasiliensisHev b 1elongation factor58P123, 124rubber (latex)Hev b 21,3-glucanase34/36C125Hev b 324P126, 127Hev b 4component of100-P128microhelix complex115 Hev b 516CU42640Hev b 6.01hevein precursor20CM36986, p02877Hev b 6.02hevein 5CM36986, p02877Hev b 6.03C-terminal fragment14CM36986, p02877Hev b 7.01hom: patatin from B-42CU80598serumHev b 7.02hom: patatin from C-44CAJ223038serumHev b 8profilin14CY15042, AJ132397,AF119365,AF1119366Hev b 9enolase51CAJ132580Hev b 10Mn superoxide dismut.26CAJ249148Hev b 11wclass 1 chitinaseCAJ238579Hev b 12lipid transfer protein 9.3CCtenocephalides felis felisCte f 1cat fleaCte f 2M1b27CAF231352Homo sapiensHom s 1 73*CY14314human autoallergensHom s 2 10.3*CX80909Hom s 3 20.1*CX89985Hom s 4 36*CY17711Hom s 5 42.6*CP02538aClone (C) or Protein (P) data

REFERENCES TO APPENDIX I

[0258] 1. Marsh, D. G., and L. R. Freidhoff. 1992. ALBE, an allergen database. IUIS, Baltimore, Md., Edition 1.0.

[0259] 2. Marsh, D. G., L. Goodfriend, T. P. King, H. Lowenstein, and T. A. E. Platts-Mills. 1986. Allergen nomenclature. Bull WHO 64:767-770.

[0260] 3. King, T. P., P. S. Norman, and J. T. Cornell. 1964. Isolation and characterization of allergen from ragweed pollen. II. Biochemistry 3:458-468.

[0261] 4. Lowenstein, H. 1980. Timothypollen allergens. Allergy 35:188-191.

[0262] 5. Aukrust, L. 1980. Purification of allergens in Cladosporium herbarum. Allergy 35:206-207.

[0263] 6. Demerec, M., E. A. Adelberg, A. J. Clark, and P. E. Hartman. 1966. A proposal for a uniform nomenclature in bacterial genetics. Genetics 54:61-75.

[0264] 7. Bodmer, J. G., E. D. Albert, W. F. Bodmer, B. Dupont, H. A. Erlich, B. Mach, S. G. E. Marsh, W. R. Mayr, P. Parham, T. Sasuki, G. M. Th. Schreuder, J. L. Strominger, A. Svejgaard, and P. I. Terasaki. 1991. Nomenclature for factors of the HLA system, 1990. hnmunogenetics 33:301-309.

[0265] 8. Griffith, I. J., J. Pollock, D. G. Klapper, B. L. Rogers, and A. K. Nault. 1991. Sequence polymorphism of Amb a I and Amb a II, the major allergens in Ambrosia artemisiifolia (short ragweed). Int. Arch. Allergy Appl. Immunol. 96:296-304.

[0266] 9. Roebber, M., D. G. Klapper, L. Goodfriend, W. B. Bias, S. H. Hsu, and D. G. Marsh. 1985. Immunochemical and genetic studies of Amb t V (Ra5G), an Ra5 homologue from giant ragweed pollen. J. Immunol. 134:3062-3069.

[0267] 10. Metzler, W. J., K. Valentine, M. Roebber, M. Friedrichs, D. G. Marsh, and L. Mueller. 1992. Solution structures of ragweed allergen Amb t V. Biochemistry 31:5117-5127.

[0268] 11. Metzler, W. J., K. Valentine, M. Roebber, D. G. Marsh, and L. Mueller. 1992. Proton resonance assignments and three-dimensional solution structure of the ragweed allergen Amb a V by nuclear magnetic resonance spectroscopy. Biochemistry 31:8697-8705.

[0269] 12. Goodfriend, L., A. M. Choudhury, J. Del Carpio, and T. P. King. 1979. Cytochromes C: New ragweed pollen allergens. Fed. Proc. 38:1415.

[0270] 13. Ekramoddoullah, A. K. M., F. T. Kisil, and A. H. Sehon. 1982. Allergenic cross reactivity of cytochrome c from Kentucky bluegrass and perennial ryegrass pollens. Mol. Immunol. 19:1527-1534.

[0271] 14. Ansari, A. A., E. A. Killoran, and D. G. Marsh. 1987. An investigation of human response to perennial ryegrass (Lolium perenne) pollen cytochrome c (Lol p X). J. Allergy Clin. Immunol. 80:229-235.

[0272] 15. Morgenstern, J. P., I. J. Griffith, A. W. Brauer, B. L. Rogers, J. F. Bond, M. D. Chapman, and M. Kuo. 1991. Amino acid sequence of Fel d I, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning. Proc. Natl. Acad. Sci. USA 88:9690-9694.

[0273] 16. Griffith, I. J., S. Craig, J. Pollock, X. Yu, J. P. Morgenstern, and B. L. Rogers. 1992. Expression and genomic structure of the genes encoding FdI, the major allergen from the domestic cat. Gene 113:263-268.

[0274] 17. Weber, A., L. Marz, and F. Altmann. 1986. Characteristics of the asparagine-linked oligosaccharide from honey-bee venom phospholipase A2. Comp. Biochem. Physiol. 83B:321-324.

[0275] 18. Weber, A., H. Schroder, K. Thalberg, and L. Marz. 1987. Specific interaction of IgE antibodies with a carbohydrate epitope of honey bee venom phospholipase A2. Allergy 42:464-470.

[0276] 19. Stanworth, D. R., K. J. Dorrington, T. E. Hugli, K. Reid, and M. W. Turner. 1990. Nomenclature for synthetic peptides representative of immunoglobulin chain sequences. Bulletin WHO 68:109-111.

[0277] 20. Rafnar, T., I. J. Griffith, M. C. Kuo, J. F. Bond, B. L. Rogers, and D. G. Klapper. 1991. Cloning of Amb a I (Antigen E), the major allergen family of short ragweed pollen. J. Biol. Chem. 266: 1229-1236.

[0278] 21. Rogers, B. L., J. P. Morgenstern, I. J. Griffith, X. B. Yu, C. M. Counsell, A. W. Brauer, T. P. King, R. D. Garman, and M. C. Kuo. 1991. Complete sequence of the allergen Amb a II: recombinant expression and reactivity with T cells from ragweed allergic patients. J. Immunol. 147:2547-2552.

[0279] 22. Klapper, D. G., L. Goodfriend, and J. D. Capra. 1980. Amino acid sequence of ragweed allergen Ra3. Biochemistry 19:5729-5734.

[0280] 23. Ghosh, B., M. P. Perry, T. Rafnar, and D. G. Marsh. 1993. Cloning and expression of immunologically active recombinant Amb a V allergen of short ragweed (Ambrosia artemisiifolia) pollen. J. Immunol. 150:5391-5399.

[0281] 24. Roebber, M., R. Hussain, D. G. Klapper, and D. G. Marsh. 1983. Isolation and properties of a new short ragweed pollen allergen, Ra6. J. Immunol. 131:706-711.

[0282] 25. Lubahn, B., and D. G. Klapper. 1993. Cloning and characterization of ragweed allergen Amb a VI (abst). J. Allergy Clin. Immunol. 91:338.

[0283] 26. Roebber, M., and D. G. Marsh. 1991. Isolation and characterization of allergen Amb a VII from short ragweed pollen. J. Allergy Clin. Immunol. 87:324.

[0284] 27. Rogers, B. L., J. Pollock, D. G. Klapper, and I. J. Griffith. 1993. Cloning, complete sequence, and recombinant expression of a novel allergen from short ragweed pollen (abst). J. Allergy Clin. Immunol. 91:339.

[0285] 28. Goodfriend, L., A. M. Choudhury, D. G. Klapper, K. M. Coulter, G. Dorval, J. DelCarpio, and C. K. Osterland. 1985. Ra5G, a homologue of Ra5 in giant ragweed pollen: isolation, HLA-DR-associated activity and amino acid sequence. Mol. Immunol. 22:899-906.

[0286] 28A. Breitenbach M, pers. comm.

[0287] 29. Nilsen, B. M., K. Sletten, M. O'Neill, B. Smestead Paulsen, and H. van Halbeek. 1991. Structural analysis of the glycoprotein allergen Art v II from pollen of mugwort (Artemesia vulgaris). J. Biol. Chem. 266:2660-2668.

[0288] 29A Jimenez A, Moreno C, Martinez J, Martinez A, Bartolome B, Guerra F, Palacios R 1994. Sensitization to sunflower pollen: only an occupational allergy? Int Arch Allergy Immunol 105:297-307.

[0289] 29B. Carnés J, Fernández-Caldas E, Casanovas M, Lahoz C, Colás C. Immunochemical characterization of Salsola kali pollen extracts. Allergy 56, Supplement 68: 274, 2001.

[0290] 29C. Giuliani A, Pini C, Bonini S, Mucci N, Ferroni L, Vicari G: Isolation and purification of a major allergen from Parietaria officinalis pollen. Allergy 42: 434-440, 1987.

[0291] 30. Smith, P. M., Suphioglu, C., Griffith, I. J., Theriault, K., Knox, R. B. and Singh, M. B. 1996. Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen. J. Allergy Clin. Immunol. 98:331-343.

[0292] 31. Suphioglu, C., Ferreira, F. and Knox, R. B. 1997. Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium-binding allergen from Bermuda grass pollen. FEBS Lett. 402:167-172.

[0293] 31 a. Asturias J A, Arilla M C, Gomez-Bayon N, Martinez J, Martinez A, and Palacios R. 1997. Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen. Clin Exp Allergy 27:1307-1313.

[0294] 32. Mecheri, S., G. Peltre, and B. David. 1985. Purification and characterization of a major allergen from Dactylis glomerata pollen: The Ag Dg 1. Int. Arch. Allergy Appl. Immunol. 78:283-289.

[0295] 33. Roberts, A. M., L. J. Bevan, P. S. Flora, I. Jepson, and M. R. Walker. 1993. Nucleotide sequence of cDNA encoding the Group II allergen of Cocksfoot/Orchard grass (Dactylis glomerata), Dac g II. Allergy 48:615-623.

[0296] 33a. Guerin-Marchand, C., Senechal, H., Bouin, A. P., Leduc-Brodard, V., Taudou, G., Weyer, A., Peltre, G. and David, B. 1996. Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen. Mol. Immunol. 33:797-806.

[0297] 34. Klysner, S., K. Welinder, H. Lowenstein, and F. Matthiesen. 1992. Group V allergens in grass pollen IV. Similarities in amino acid compositions and amino terminal sequences of the group V allergens from Lolium perenne, Poa pratensis and Dactylis glomerata. Clin. Exp. Allergy 22: 491-497.

[0298] 35. Perez, M., G. Y. Ishioka, L. E. Walker, and R. W. Chesnut. 1990. cDNA cloning and immunological characterization of the rye grass allergen Lol p I. J. Biol. Chem. 265:16210-16215.

[0299] 36. Griffith, 1. J., P. M. Smith, J. Pollock, P. Theerakulpisut, A. Avjioglu, S. Davies, T. Hough, M. B. Singh, R. J. Simpson, L. D. Ward, and R. B. Knox. 1991. Cloning and sequencing of Lol p I, the major allergenic protein of rye-grass pollen. FEBS Letters 279:210-215.

[0300] 37. Ansari, A. A., P. Shenbagamurthi, and D. G. Marsh. 1989. Complete amino acid sequence of a Lolium perenne (perennial rye grass) pollen allergen, Lol p 11. J. Biol. Chem. 264:11181-11185.

[0301] 37a. Sidoli, A., Tamborini, E., Giuntini, I., Levi, S., Volonte, G., Paini, C., De Lalla, C., Siccardi, A. G., Baralle, F. E., Galliani, S. and Arosio, P. 1993. Cloning, expression, and immunological characterization of recombinant Lolium perenne allergen Lol p 11. J. Biol. Chem. 268:21819-21825.

[0302] 38. Ansari, A. A., P. Shenbagamurthi, and D. G. Marsh. 1989. Complete primary structure of a Lolium perenne (perennial rye grass) pollen allergen, Lol p II: Comparison with known Lol p I and II sequences. Biochemistry 28:8665-8670.

[0303] 39. Singh, M. B., T. Hough, P. Theerakulpisut, A. Avjioglu, S. Davies, P. M. Smith, P. Taylor, R. J. Simpson, L. D. Ward, J. McCluskey, R. Puy, and R. B. Knox. 1991. Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: Intracellular targeting to the amyloplost. Proc. Natl. Acad. Sci. 88:1384-1388.

[0304] 39a. van Ree R, Hoffinan D R, van Dijk W, Brodard V, Mahieu K, Koeleman C A, Grande M, van Leeuwen W A, Aalberse R C. 1995. Lol p XI, a new major grass pollen allergen, is a member of a family of soybean trypsin inhibitor-related proteins. J Allergy Clin Immunol 95:970-978.

[0305] 40. Suphioglu, C. and Singh, M. B. 1995. Cloning, sequencing and expression in Escherichia coli of Pha a 1 and four isoforms of Pha a 5, the major allergens of canary grass pollen. Clin. Exp. Allergy 25:853-865.

[0306] 41. Dolecek, C., Vrtala, S., Laffer, S., Steinberger, P., Kraft, D., Scheiner, O. and Valenta, R. 1993. Molecular characterization of Phl p II, a major timothy grass (Phleum pratense) pollen allergen. FEBS Lett. 335:299-304.

[0307] 41A. Fischer S, Grote M, Fahlbusch B, Muller WD, Kraft D, Valenta R. 1996. Characterization of Phl p 4, a major timothy grass (Phleum pratense) pollen allergen. J Allergy Clin Immunol 98:189-198.

[0308] 42. Matthiesen, F., and H. Lowenstein. 1991. Group V allergens in grass pollens. I. Purification and characterization of the group V allergen from Phleum pratense pollen, Phl p V. Clin. Exp. Allergy 21:297-307.

[0309] 43. Petersen, A., Bufe, A., Schramm, G., Schlaak, M. and Becker, W. M. 1995. Characterization of the allergen group VI in timothy grass pollen (Phl p 6). II. cDNA cloning of Phl p 6 and structural comparison to grass group V. Int. Arch. Allergy Immunol. 108:55-59.

[0310] 44. Valenta, R., Ball, T., Vrtala, S., Duchene, M., Kraft, D. and Scheiner, O. 1994. cDNA cloning and expression of timothy grass (Phleum pratense) pollen profilin in Escherichia coli: comparison with birch pollen profilin. Biochem. Biophys. Res. Commun. 199:106-118.

[0311] 46. Esch, R. E., and D. G. Klapper. 1989. Isolation and characterization of a major cross-reactive grass group I allergenic determinant. Mol. Immunol. 26:557-561.

[0312] 47. Olsen, E., L. Zhang, R. D. Hill, F. T. Kisil, A. H. Sehon, and S. Mohapatra. 1991. Identification and characterization of the Poa p IX group of basic allergens of Kentucky bluegrass pollen. J. Immunol. 147:205-211.

[0313] 48. Avjioglu, A., M. Singh, and R. B. Knox. 1993. Sequence analysis of Sor h I, the group I allergen of Johnson grass pollen and it comparison to rye-grass Lol p I (abst). J. Allergy Clin. Immunol. 91:340.

[0314] 49. Breiteneder H, Pettenburger K, Bito A, Valenta R, Kraft D, Rumpold H, Scheiner O, Breitenbach M. The gene coding for themajor birch pollen allergen BetvI, is highly homologus to a pea disease resistance response gene. EMBO J. 8: 1935-1938,1989.

[0315] 50. Swoboda I, Jilek A, Ferreira F, Engel E, Hoffman-Sommergruber K, Scheiner O, Kraft D, Breiteneder H, Pittenauer E, Schmid E. Isoforms of Bet v 1, the major birch pollen allergen, analyzed by liquid chromatography, mass spectrometry, and cDNA cloning. J. Biol. Chem. 270: 2607-2613, 1995.

[0316] 51. Larsen J N, Stroman P, Ipsen H. PCR based cloning and sequencing of isogenes encoding the tree pollen major allergen Car b I from Carpinus betulus, hornbeam. Mol. Immunol. 29: 703-711, 1992.

[0317] 52. Kos T, Hoffmann-Sommergruber K, Ferreira F, Hirschwehr R, Ahom H, Horak F, Jager S, Sperr W, Kraft D, Scheiner O. 1993. Purification, characterization and N-terminal amino acid sequence of a new major allergen from European chestnut pollen—Cas s 1. Biochem Biophys Res Commun 196:1086-92.

[0318] 53. Diaz-Perales A, Lombardero M, Sánchez-Monge R, García-Sellés F J, Pernas M, Fernádndez-Rivas M, Barber D, Salcedo G. 2000. Lipid-transfer proteins as potential plant panallergens: cross-reactivity among proteins of Artemisia pollen, Castaneae nut and Rosaceae fruits, with different IgE-binding capacities. Clin Exp Allergy 30:1403-1410.

[0319] 54. Ipsen, H., and O. C. Hansen. 1991. The NH2-terminal amino acid sequence of the immunochemically partial identical major allergens of alder (Alnus glutinosa) Aln g I, birch (Betula verrucosa) Bet v I, hornbeam (Carpinus betulus) Car b I and oak (Quercus alba) Que a I pollens. Mol. Immunol. 28:1279-1288.

[0320] 55. Taniai, M., S. Ando, M. Usui, M. Kurimoto, M. Sakaguchi, S. Inouye, and T. Matuhasi. 1988. N-terminal amino acid sequence of a major allergen of Japanese cedar pollen (Cry j I). FEBS Lett. 239:329-332.

[0321] 56. Griffith, I. J., A. Lussier, R. Garman, R. Koury, H. Yeung, and J. Pollock. 1993. The cDNA cloning of Cry j I, the major allergen of Cryptomeria japonica (Japanese cedar) (abst). J. Allergy Clin. Immunol. 91:339.

[0322] 57. Sakaguchi, M., S. Inouye, M. Taniai, S. Ando, M. Usui, and T. Matuhasi. 1990. Identification of the second major allergen of Japanese cedar pollen. Allergy 45:309-312.

[0323] 57A. Yokoyama M, Miyahara M, Shimizu K, Kino K, Tsunoo H. 2000. Purification, identification, and cDNA cloning of Jun a 2, the second major allergen of mountain cedar pollen. Biochem Biophys Res Commun 275:195-202.

[0324] 57B. Midoro-Horiuti T, Goldblum R M, Kurosky A, Wood T G, Brooks E G. 2000. Variable Expression of Pathogenesis-Related Protein Allergen in Mountain Cedar (Juniperus ashei) Pollen. J Immunol 164:2188-2192.

[0325] 57C. Tinghino R., Barletta B., Palumbo S., Afferni C., lacovacci P., Mari A., Di Felice G., Pini, C. 1998. Molecular characterization of a cross-reactive Juniperus oxycedrus pollen allergen, Jun o 2: a novel calcium-binding allergen J. Allergy Clin. Immunol. 101:772-777.

[0326] 58 Gross G N, Zimburean J M, Capra J D 1978. Isolation and partial characterization of the allergen in mountain cedar pollen. Scand J Immunol 8:437-41 .

[0327] 58A Obispo T M, Melero J A, Carpizo J A, Carreira J, Lombardero M 1993. The main allergen of Olea europaea (Ole e I) is also present in other species of the oleaceae family. Clin Exp Allergy 23:311-316.

[0328] 59. Lombardero M., Barbas J. A., Moscoso del Prado J., Carreira J. 1994. cDNA sequence analysis of the main olive allergen, Ole e I. Clin. Exp. Allergy 24:765-770.

[0329] 60. Villalba, M., E. Batanero, C. Lopez-Otin, L. M. Sanchez, R. I. Monsalve, M. A. Gonzalez de la Pena, C. Lahoz, and R. Rodriguez. 1993. Amino acid sequence of Ole e I, the major allergen from olive tree pollen (Olea europaea). Eur. J. Biochem. 216:863-869.

[0330] 60A. Asturias J A, Arilla M C, Gomez-Bayon N, Martinez J, Martinez A, Palacios R 1997. Cloning and expression of the panallergen profilin and the major allergen (Ole e 1) from olive tree pollen. J Allergy Clin Immunol 100:365-372.

[0331] 60B. Batanero E, Villalba M, Ledesma A Puente XS, Rodriguez R. 1996. Ole e 3, an olive-tree allergen, belongs to a widespread family of pollen proteins. Eur J Biochem 241: 772-778.

[0332] 60C. Batanero E, Ledesma A, Villalba M, Rodriguez R. 1997. Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen. FEBS Lett. 410:293-296.

[0333] 60D. Tejera M L, Villalba M, Batanero E, Rodriguez R. 1999. Identification, isolation, and characterization of Ole e 7, a new allergen of olive tree pollen. J Allergy Clin Immunol 104:797-802.

[0334] 60E. Ledesma, A., Villalba, M. and Rodriguez, R. 2000. Cloning, expression and characterization of a novel four EF-hand Ca(2+)-binding protein from olive pollen with allergenic activity. FEBS Lett. 466:192-196.

[0335] 61. Chua, K. Y., G. A. Stewart, and W. R. Thomas. 1988. Sequence analysis of cDNA encoding for a major house dust mite allergen, Der p I. J. Exp. Med. 167:175-182.

[0336] 62. Chua, K. Y., C. R. Doyle, R. J. Simpson, K. J. Turner, G. A. Stewart, and W. R. Thomas. 1990. Isolation of cDNA coding for the major mite allergen Der p II by IgE plaque immunoassay. Int. Arch. Allergy Appl. Immunol. 91:118-123.

[0337] 63. Smith W A, Thomas W R. 1996. Comparative analysis of the genes encoding group 3 allergens from Dermatophagoides pteronyssinus and Dermatophagoides farinae. Int Arch Allergy Immunol 109:133-40.

[0338] 64. Lake, F. R., L. D. Ward, R. J. Simpson, P. J. Thompson, and G. A. Stewart. 1991. House dust mite-derived amylase: Allergenicity and physicochemical characterisation. J. Allergy Clin. Immunol. 87:1035-1042.

[0339] 65. Tovey, E. R., M. C. Johnson, A. L. Roche, G. S. Cobon, and B. A. Baldo. 1989. Cloning and sequencing of a cDNA expressing a recombinant house dust mite protein that binds human IgE and corresponds to an important low molecular weight allergen. J. Exp. Med. 170:1457-1462.

[0340] 66. Yasueda, H., T. Shida, T. Ando, S. Sugiyama, and H. Yamakawa. 1991. Allergenic and proteolytic properties of fourth allergens from Dermatophagoides mites. In: “Dust Mite Allergens and Asthma. Report of the 2nd international workshop” A. Todt, Ed., UCB Institute of Allergy, Brussels, Belgium, pp. 63-64.

[0341] 67. Shen, H. -D., K. -Y. Chua, K. -.L. Lin, K. -H. Hsieh, and W. R. Thomas. 1993. Molecular cloning of a house dust mite allergen with common antibody binding specificities with multiple components in mite extracts. Clin. Exp. Allergy 23:934-40.

[0342] 67A. O'Neil G M, Donovan G R, Baldo B A. 1994. Cloning and charaterisation of a major allergen of the house dust mite Dermatophagoides pteronyssinus, homologous with glutathione S-transferase. Biochim Biophys Acta, 1219:521-528.

[0343] 67B. King C, Simpson R J, Moritz R L, Reed G E, Thompson P J, Stewart G A. 1996. The isolation and characterization of a novel collagenolytic serine protease allergen (Der p 9) from the dust mite Dermatophagoides pteronyssinus. J Allergy Clin Immunol 98:739-47.

[0344] 68. Lind P, Hansen OC, Horn N. 1988. The binding of mouse hybridoma and human IgE antibodies to the major fecal allergen, Der p I of D. pteronyssinus. J. Immunol. 140:4256-4262.

[0345] 69. Dilworth, R. J., K. Y. Chua, and W. R. Thomas. 1991. Sequence analysis of cDNA coding for a mojor house dust allergn Der f I. Clin. Exp. Allergy 21:25-32.

[0346] 70. Nishiyama, C., T. Yunki, T. Takai, Y. Okumura, and H. Okudaira. 1993. Determination of three disulfide bonds in a major house dust mite allergen, Der f II. Int. Arch. Allergy Immunol. 101:159-166.

[0347] 71. Trudinger, M., K. Y. Chua, and W. R. Thomas. 1991. cDNA encoding the major dust mite allergen Der f II. Clin. Exp. Allergy 21:33-38.

[0348] 71A. Tategaki A, Kawamoto S, Aki T, Jyo T, Suzuki O, Shigeta S, Ono K. Newly described house dust mite allergens. ACI International suppl. 1: 74-76, 2000.

[0349] 72. Aki T, Kodama T, Fujikawa A, Miura K, Shigeta S, Wada T, Jyo T, Murooka Y, Oka S, Ono K. 1995. Immunochemical characteristion of recombinant and native tropomyosins as a new allergen from the house dust mite Dermatophagoides farinae. J Allergy Clin Immunol 96:74-83.

[0350] 72A. Tsai L, Sun Y, Chao P, Ng H, Hung M, Hsieh K, Liaw S, Chua K, 1999. Sequence analysis and expression of a cDNA clone encoding a 98-kDa allergen in Dermatophagoides farinae. Clin Exp Allergy 29:1606-1613.

[0351] 72B. Gafvelin G, Johansson E, Lundin A, Smith A M, Chapman M D, Benjamin D C, Derewenda U, Van Hage-Hamsten M. 2001. Cross-reactivity studies of a new group 2 allergen from the dust mite Glycyphagus domesticus, Gly d 2, and group 2 allergens from Dermatophagoides pteronyssinus, Lepidoglyphus destructor, and Tyrophagus putrescentiae with recombinant allergens. J Allergy Clin Immunol 107:511-518.

[0352] 73. van Hage-Hamsten, M., T. Bergman, E. Johansson, B. Persson, H. Jomvall, B. Harfast, and S. G. O. Johansson. 1993. N-terminal amino acid sequence of major allergen of the mite lepidoglyphus destructor (abst). J. Allergy Clin. Immunol. 91:353.

[0353] 74. Varela J, Ventas P, Carreira J, Barbas JA, Gimenez-Gallego G, Polo F. Primary structure of Lep d I, the main Lepidoglyphus destructor allergen. Eur J Biochem 225:93-98, 1994.

[0354] 75. Schmidt M, van der Ploeg I, Olsson S, van Hage Hamsten M. The complete cDNA encoding the Lepidoglyphus destructor major allergen Lep d 1. FEBS Lett 370:11-14, 1995.

[0355] 75A. Eriksson T L J, Rasool O, Huecas S, Whitley P, Crameri R, Appenzeller U, Gafvelin G, van Hage-Hamsten M. 2001. Cloning of three new allergens from the dust mite Lepidoglyphus destructor using phage surface display technology. Eur. J. Biochem. 268:287-294.

[0356] 75B. Eriksson T L, Johansson E, Whitley P, Schmidt M, Elsayed S, van Hage-Hamsten M. 1998. Cloning and characterisation of a group II allergen from the dust mite Tyrophagus putrescentiae. Eur. J. Biochem. 251 (1-2), 443-447.

[0357] 76. Rautiainen J, Rytkonen M, Pelkonen J, Pentikainen J, Perola O, Virtanen T, Zeiler T, Mantyarvi R. BDA20, a major bovine dander allergen characterized at the sequence level is Bos d 2. Submitted.

[0358] 77. Gjesing B, Lowenstein H. Immunochemistry of food antigens. Ann Allergy 53:602, 1984.

[0359] 78. de Groot, H., K. G. H. Goei, P. van Swieten, and R. C. Aalberse. 1991. Affinity purification of a major and a minor allergen from dog extract: Serologic activity of afflity-purified Can f I and Can f I-depleted extract. J. Allergy Clin. Immunol. 87:1056-1065.

[0360] 79. Konieczny, A. Personal communication; Immunologic Pharmaceutical Corp.

[0361] 79A. Bulone, V. 1998. Separation of horse dander allergen proteins by two-dimensional electrophoresis. Molecular characterisation and identification of Equ c 2.0101 and Equ c 2.0102 as lipocalin proteins. Eur J Biochem 253:202-211.

[0362] 79B. Swiss-Prot acc. P81216, P81217.

[0363] 79C. Dandeu J. P., Rabillon J., Divanovic A., Carmi-Leroy A., David B. (1993). Hydrophobic interaction chromatography for isolation and purification of Equ c 1, the horse major allergen. J. Chromatogr. 621:23-31.

[0364] 79D. Goubran Botros H., Rabillon J., Gregoire C., David B., Dandeu J. P. 1998. Thiophilic absorption chromatography: purification of Equ c 2 and Equ c 3, two horse allergens from horse sweat. J. Chromatogr. B 710:57-65.

[0365] 79E. Hilger C, Kohnen M, Grigioni F, Lehners C, Hentges F. Allergic cross-reactions between cat and pig serum albumin. Allergy 52:179-187, 1997; and Hilger C, Grigioni F, Hentges F. Sequence of the gene encoding cat (Felis domesticus) serum albumin. Gene 169:295-296, 1996.

[0366] 79F. Ichikawa K, Vailes L D, Pomes A, Chapman M D. Molecular cloning, expression and modeling of cat allergen, cystatin (Fel d 3), a cysteine protease inhibitor. Clin Exp Allergy, In Press 2001.

[0367] 80. McDonald, B., M. C. Kuo, J. L. Ohman, and L. J. Rosenwasser. 1988. A 29 amino acid peptide derived from rat alpha 2 euglobulin triggers murine allergen specific human T cells (abst). J. Allergy Clin. Immunol. 83:251.

[0368] 81. Clarke, A. J., P. M. Cissold, R. A. Shawi, P. Beattie, and J. Bishop. 1984. Structure of mouse urinary protein genes: differential splicing configurations in the 3′-non-coding region. EMBO J 3:1045-1052.

[0369] 82. Longbottom, J. L. 1983. Chracterization of allergens from the urines of experimental animals. McMillan Press, London, pp. 525-529.

[0370] 83. Laperche, Y., K. R. Lynch, K. P. Dolans, and P. Feigelsen. 1983. Tissue-specific control of alpha 2u globulin gene expression: constitutive synthesis in submaxillary gland. Cell 32:453-460.

[0371] 83A. Bush R K, Sanchez H, Geisler D. 1999. Molecular cloning of a major Altemaria alternata allergen, rAlt a 2. J Allergy Clin Immunol 104:665-671.

[0372] 83B. Aukrust L, Borch S M. 1979. Partial purification and characterization of two Cladosporium herbarum allergens. Int Arch Allergy Appl Immunol 60:68-79.

[0373] 83C. Sward-Nordino M, Paulsen B S, Wold J K. 1988. The glycoprotein allergen Ag-54 (Cla h II) from Cladosporium herbarum. Structural studies of the carbohydrate moiety. Int Arch Allergy Appl Immunol 85:288-294.

[0374] 84. Shen, et al. J. Allergy Clin. Immunol. 103:S157, 1999.

[0375] 84A. Crameri R. Epidemiology and molecular basis of the involvement of Aspergillus fumigatus in allergic diseases. Contrib. Microbiol. Vol. 2, Karger, Basel (in press).

[0376] 84B. Shen, et al. (manuscript submitted), 1999

[0377] 84C. Shen H D, Ling W L, Tan M F, Wang S R, Chou H, Han S I H. Vacuolar serine proteinase: A major allergen of Aspergillus fumigatus. 10th International Congress of Immunology, Abstract, 1998.

[0378] 85. Kumar, A., L. V. Reddy, A. Sochanik, and V. P. Kurup. 1993. Isolation and characterization of a recombinant heat shock protein of Aspergillus fumigatus. J. Allergy Clin. Immunol. 91:1024-1030.

[0379] 86A. Shen H D, Lin W L, Tsai J J, Liaw S F, Han S H. 1996. Allergenic components in three different species of Penicillium: crossreactivity among major allergens. Clin Exp Allergy 26:444-451.

[0380] 86B. Shen, et al. Abstract; The XVIII Congress of the European Academy of Allergology and Clinical Immunology, Brussels, Belgium, 3-7 July 1999.

[0381] 87. Shen H D, Liaw S F, Lin W L, Ro L H, Yang H L, Han S H. 1995. Molecular cloning of cDNA coding for the 68 kDa allergen of Penicillium notatum using MoAbs. Clin Exp Allergy 25:350-356.

[0382] 88. Shen, H. D., K. B. Choo, H. H. Lee, J. C. Hsieh, and S. H. Han. 1991. The 40 kd allergen of Candida albicans is an alcohol dehydrogenease: molecular cloning and immunological analysis using monoclonal antibodies. Clin. Exp. Allergy 21:675-681.

[0383] 89. Shen, et al. Clin. Exp. Allergy (in press), 1999.

[0384] 90. Woodfolk J A, Wheatley L M, Piyasena R V, Benjamin D C, Platts-Mills T A.1998. Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity. Sequence homology to two families of serine proteinases. J Biol Chem 273:29489-96.

[0385] 91. Deuell, B., L. K. Arruda, M. L. Hayden, M. D. Chapman and T. A. E. Platts-Mills. 1991. Trichophyton tonsurans Allergen I. J. Immunol. 147:96-101.

[0386] 91A. Schmidt M, Zargari A, Holt P, Lindbom L, Hellnan U, Whitley P, van der Ploeg I, Harfast B, Scheynius A. 1997. The complete cDNA sequence and expression of the first major allergenic protein of Malassezia furfur, Mal f 1. Eur J Biochem 246:181-185.

[0387] 91B. Homer WE, Reese G, Lehrer SB. 1995. Identification of the allergen Psi c 2 from the basidiomycete Psilocybe cubensis as a fungal cyclophilin. Int Arch Allergy Immunol 107:298-300.

[0388] 91C. Hoffman D R, Schmidt J O. 2000. Phospholipases from Asian honeybees. J Allergy Clin Immunol 105:S56 (abs).

[0389] 92. Kuchler, K., M. Gmachl, M. J. Sippl, and G. Kreil. 1989. Analysis of the cDNA for phospholipase A2 from honey bee venom glands: The deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes. Eur. J. Biochem. 184:249-254.

[0390] 93. Gmachl, M., and G. Kreil. 1993. Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm. Proc. Natl. Acad. Sci. USA 90:3569-3573.

[0391] 93A. Hoffman DR. 1977. Allergens in bee venom III. Identification of allergen B as an acid phosphatase. J Allergy Clin. Immunol. 59:364-366.

[0392] 94. Habermann, E. 1972. Bee and wasp venoms. Science 177:314-322.

[0393] 95. Hoffman D R, Jacobson R S. 1996. Allergens in Hymenoptera venom XXVII: Bumblebee venom allergy and allergens. J. Allergy Clin. Immunol. 97:812-821.

[0394] 95A. Hoffman DR, El-Choufani A E, Smith M M, de Groot H. 2001. Occupational allergy to bumblebee venom: Allergens of Bombus terrestris. J Allergy Clin Immunol In press.

[0395] 95B. Helm R, Cockrell G, Stanley J S, Brenner R J, Burks W, Bannon G A. 1996. Isolation and characterization of a clone encoding a majore allergen (Bla g Bd90K) involved in IgE mediated cockroach hypersensitivity. J Allerg Clin Immunol 98:172-180.

[0396] 95C. Pomes A, Melen E, Vailes L D, Retief J D, Arruda L K, Chapman M D. 1998. Novel allergen structures with tandem amino acid repeats derived from German and American cockroach. J Biol Chem 273:30801-30807.

[0397] 96. Arruda L K, Vailes L D, Mann B J, Shannon J, Fox J W, Vedvick T S, Hayden M L, Chapman M D. Molecular cloning of a major cockroach (Blattella germanica) allergen, Bla g 2. Sequence homology to the aspartic proteases. J Biol Chem 270:19563-19568, 1995.

[0398] 97. Arruda L K, Vailes L D, Hayden M L, Benjamin D C, Chapman M D. Cloning of cockroach allergen, Bla g 4, identifies ligand binding proteins (or calycins) as a cause of IgE antibody responses. J Biol Chem 270:31196-31201, 1995.

[0399] 98. Arruda L K, Vailes L D, Benjamin D C, Chapman M D. Molecular cloning of German Cockroach (Blattella germanica) allergens. Int Arch Allergy Immunol 107:295-297, 1995.

[0400] 98A. Wu C H, Wang N M, Lee M F, Kao CYY, Luo S F. 1998. Cloning of the American cockroach Cr-PII allergens: Evidence for the existence of cross-reactive allergens between species. J Allergy Clin Immunol 101:832-840.

[0401] 98B. Melen E, Pomes A, Vailes L D, Arruda L K, Chapman M D. 1999. Molecular cloning of Per a 1 and definition of the cross-reactive Group 1 cockroach allergens. J Allergy Clin Immunol 103:859-64.

[0402] 98C. Wu C H, Lee M F, Liao S C, Luo S F. Sequencing analysis of cDNA clones encoding the American cockroach Cr-PI allergens. J Biol Chem 271:17937-17943, 1996.

[0403] 98D. Wu C H, Lee M F, Wang N M, Luo S F. Sequencing and immunochemical characterization of the American cockroach Per a 3 (Cr-PI) isoallergenic variants. Molecular Immunol 34:1-8, 1997.

[0404] 98E. Santos A B R, Chapman M D, Aalberse R C, Vailes LD, Ferriani V P L, Oliver C, Rizzo M C, Naspitz C K, Arruda L K. 1999. Cockroach allergens and asthma in Brazil: Identification of tropomyosin as a major allergen with potential cross-reactivity with mite and shrimp allergens. J Allergy Clin Immunol 104:329-337.

[0405] 98F. Asturias J A, Gómez-Bayón, Arilla M C, Martinez A, Palacios R, Sánchez-Gascón, Martinez J. 1999. Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen. J Immunol 162:4342-4348.

[0406] 99. Mazur, G., X. Baur, and V. Liebers. 1990. Hypersensitivity to hemoglobins of the Diptera family Chironomidae: Structural and functional studies of their immunogenic/allergenic sites. Monog. Allergy 28:121-137.

[0407] 100. Soldatova, L., L. Kochoumian, and T. P. King. 1993. Sequence similarity of a hornet (D. maculata) venom allergen phospholipase Al with mammalian lipases. FEBS Letters 320:145-149.

[0408] 101. Lu, G., L. Kochoumian and T. P. King. Whiteface hornet venom allergen hyaluronidase: cloning and its sequence similarity with other proteins (abst.). 1994. J. Allergy Clin. Immunol. 93:224.

[0409] 102. Fang, K. S. F., M. Vitale, P. Fehlner, and T. P. King. 1988. cDNA cloning and primary structure of a white-faced hornet venom allergen, antigen 5. Proc. Natl. Acad. Sci., USA 85:895-899.

[0410] 103. King, T. P., D. C. Moran, D. F. Wang, L. Kochoumian, and B. T. Chait. 1990. Structural studies of a hornet venom allergen antigen 5, Dol m V and its sequence similarity with other proteins. Prot. Seq. Data Anal. 3:263-266.

[0411] 104. Lu, G., M. Villalba, M. R. Coscia, D. R. Hoffman, and T. P. King. 1993. Sequence analysis and antigen cross reactivity of a venom allergen antigen 5 from hornets, wasps and yellowjackets. J. Immunol. 150: 2823-2830.

[0412] 105. King, T. P. and Lu, G. 1997. Unpublished data.

[0413] 105A. King T P, Lu G, Gonzalez M, Qian N and Soldatova L. 1996. Yellow jacket venom allergens, hyaluronidase and phospholipase: sequence similarity and antigenic cross-reactivity with their hornet and wasp homologs and possible implications for clinical allergy. J. Allergy Clin. Immunol. 98:588-600.

[0414] 106. Hoffman, D. R. 1993. Allergens in hymenoptera venom XXV: The amino acid sequences of antigen 5 molecules and the structural basis of antigenic cross-reactivity. J. Allergy Clin. Immunol. 92:707-716.

[0415] 107. Hoffman, D. R. 1992. Unpublished data.

[0416] 108. Hoffman, D. R. 1993. The complete amino acid sequence of a yellowjacket venom phospholipase (abst). J. Allergy Clin. Immunol. 91:187.

[0417] 109. Jacobson, R. S., D. R. Hoffman, and D. M. Kemeny. 1992. The cross-reactivity between bee and vespid hyaluronidases has a structural basis (abst). J. Allergy Clin. Immunol. 89:292.

[0418] 110. Hoffman, D. R. 1993. Allergens in Hymenoptera venom XXIV: The amino acid sequences of imported fire ant venom allergens Sol i II, Sol i III, and Sol i IV. J. Allergy Clin. Immunol. 91:71-78.

[0419] 111. Schmidt, M., R. B. Walker, D. R. Hoffman, and T. J. McConnell. 1993. Nucleotide sequence of cDNA encoding the fire ant venom protein Sol i II. FEBS Letters 319:138-140.

[0420] 112. Elsayed S, Bennich H. The primary structure of Allergen M from cod. Scand J Immunol 3:683-686, 1974.

[0421] 113. Elsayed S, Aas K, Sletten K, Johansson SGO. Tryptic cleavage of a homogeneous cod fish allergen and isolation of two active polypeptide fragments. Immunochemistry 9:647-661, 1972.

[0422] 114. Hoffman, D. R. 1983. Immunochemical identification of the allergens in egg white. J. Allergy Clin. Immunol. 71:481-486.

[0423] 115. Langeland, T. 1983. A clinical and immunological study of allergy to hen's egg white. IV. specific IgE antibodies to individual allergens in hen's egg white related to clinical and immunolgical parameters in egg-allergic patients. Allergy 38:493-500.

[0424] 116. Daul, C. B., M. Slattery, J. E. Morgan, and S. B. Lehrer. 1993. Common crustacea allergens: identification of B cell epitopes with the shrimp specific monoclonal antibodies. In: “Molecular Biology and Immunology of Allergens” (D. Kraft and A. Sehon, eds.). CRC Press, Boca Raton. pp.291-293.

[0425] 117. K. N. Shanti, B. M. Martin, S. Nagpal, D. D. Metcalfe, P. V. Subba Rao. 1993. Identification of tropomyosin as the major shrimp allergen and characterization of its IgE-binding epitopes. J. Immunol. 151:5354-5363.

[0426] 117A. M. Miyazawa, H. Fukamachi, Y. Inagaki, G. Reese, C. B. Daul, S. B. Lehrer, S. Inouye, M. Sakaguchi. 1996. Identification of the first major allergen of a squid (Todarodes pacificus). J. Allergy Clin. Immunol. 98:948-953.

[0427] 117B Lopata A L, Zinn C, Potter P C. Characteristics of hypersensitivity reactions and identification of a unique 49 kd IgE-binding protein (Hal-m-1) in abalone (Haliotis midae). J. Allergy Clin. Immunol. 100: 642-648, 1997.

[0428] 117C. K. Hoffmann-Sommergruber, G. O'Riordain, H. Ahom, C. Ebner, M. Laimer Da Camara Machado, H. Puhringer, O. Scheiner, H. Breiteneder. 1999. Molecular characterization of Dau c 1, the Bet v 1 homologous protein from carrot and its cross-reactivity with Bet v 1 and Api g 1 Clin Exp Allergy 29:840-847.

[0429] 118. Monsalve, R. I., M. A. Gonzalez de la Pena, L. Menendez-Arias, C. Lopez-Otin, M. Villalba, and R. Rodriguez. 1993. Characterization of a new mustard allergen, Bra j IE. Detection of an allergenic epitope. Biochem. J. 293:625-632.

[0430] 118A. Monsalve R I, Gonzalez de la Pena M A, Lopez-Otin C, Fiandor A, Fernandez C, Villalba M, Rodriguez R. 1997. Detection, isolation and complete amino acid sequence of an aeroallergenic protein from rapeseed flour. Clin Exp Allergy 27:833-841.

[0431] 119. Mena, M., R. Sanchez?Monge, L. Gomez, G. Salcedo, and P. Carbonero. 1992. A major barley allergen associated with baker's asthma disease is a glycosylated monomeric inhibitor of insect alpha-amylase: cDNA cloning and chromosomal location of the gene. Plant Molec. Biol. 20:451-458.

[0432] 119A. Xu H, Theerakulpisut P, Goulding N, Suphioglu C, Singh M. B. Bhalla P. L. Cloning expression and immunological characterization of Ory s 1, the major allergen of rice pollen. Gene 164:255-259, 1995.

[0433] 119B. Pastorello E A, Ortolani C, Farioli L, Pravettoni V, Ispano M, Borga A, Bengtsson A, Incorvaia C, Berti C, Zanussi C. 1994. Allergenic cross-reactivity among peach, apricot, plum, and cherry in patients with oral allergy syndrome: an in vivo and in vitro study. J. Allergy Clin. Immunol. 94:699-707.

[0434] 120. Menendez-Arias, L., I. Moneo, J. Dominguez, and R. Rodriguez. 1988. Primary structure of the major allergen of yellow mustard (Sinapis alba L.) seed, Sin a I. Eur. J. Biochem. 177:159-166.

[0435] 121. Gonzalez R, Varela J, Carreira J, Polo F. Soybean hydrophobic protein and soybean hull allergy. Lancet 346:48-49, 1995.

[0436] 121A. Pastorello E A, Varin E, Farioli L, Pravettoni V, Ortolani C, Trambaioli C, Fortunato D, Giuffrida MG, Rivolta F, Robino A, Calamari A M, Lacava L, Conti A. The major allergen of sesame seeds (Sesamum indicum) is a 2S albumin. J. Chromatogr. B Biomed. Sci. Appl. 756: 85-93, 2001.

[0437] 121B. Moneo I, Caballero M L, Gomez F, Ortega E, Alonso M J. Isolation and characterization of a major allergen from the fish parasite Anisakis simplex. J. Allergy Clin. Immunol. 106: 177-182, 2000.

[0438] 121C. Asturias JA, Eraso E, Martinez A. 2000. Is tropomysoin an allergen in Anisakis? Allergy 55:898-890.

[0439] 122. Christie, J. F., B. Dunbar, I. Davidson, and M. W. Kennedy. 1990. N-terminal amino acid sequence identity between a major allergen of Ascaris lumbricoides and Ascaris suum and MHC-restricted IgE responses to it. Immunology 69:596-602.

[0440] 122A. Onizuka R, Kamiya H, Muramoto K, Goto R, Inoue K, Kumamoto K, Nakajima Y, Iida S, Ishigami F. Purification of major allergens from red soft coral (Dendrophythya nipponica). Int Arch Allergy Immunol, In press 2001.

[0441] 123. Czuppon A B, Chen Z, Rennert S, Engelke T, Meyer H E, Heber M, Baur X. The rubber elongation factor of rubber trees (Hevea brasiliensis) is the major allergen in latex. J Allergy Clin Immunol 92:690-697, 1993.

[0442] 124. Attanayaka DPSTG, Kekwick R G O, Franklin F C H. 1991. Molecular cloning and nucleotide sequencing of the rubber elongation factor gene from hevea brasiliensis. Plant Mol Biol 16:1079-1081.

[0443] 125. Chye M L, Cheung K Y. 1995. J 1,3-glucanase is highly expressed in Laticifers of Hevea brasiliensis. Plant Mol Biol 26:397-402.

[0444] 126. Alenius H, Palosuo T, Kelly K, Kurup V, Reunala T, Makinen-Kiljunen S, Turjanmaa K Fink J. 1993. IgE reactivity to 14-kD and 27-kD natural rubber proteins in Latex-allergic children with Spina bifida and other congenital anomalies. Int Arch Allergy Immunol 102:61-66.

[0445] 127. Yeang H Y, Cheong K F, Sunderasan E, Hamzah S, Chew N P, Hamid S, Hamilton R G, Cardosa M J. 1996. The 14.6 kD (REF, Hev b 1) and 24 kD (Hev b 3) rubber particle proteins are recognized by IgE from Spina Bifida patients with Latex allergy. J Allerg Clin Immunol in press.

[0446] 128. Sunderasan E, Hamzah S, Hamid S, Ward M A, Yeang H Y, Cardosa M J. 1995. Latex B-serum J-1,3-glucanase (Hev b 2) and a component of the microhelix (Hev b 4) are major Latex allergens. J nat Rubb Res 10:82-99.

13TABLE 9Selected allergens with structures available in Protein Database (PDB)ID NO: 1A0KDeposited: 02 Dec. 1997 Exp. Method: X-ray Diffraction Resolution: 2.20 ÅTitleProfilin I From Arabidopsis ThalianaClassificationCytoskeletonCompoundMol_Id: 1; Molecule: Profilin; Chain: Null; Engineered: Recombinant PlantProtein; Biological_Unit: MonomerID NO: 1A9VDeposited: 10 April 1998 Exp. Method: NMR, 10 StructuresTitleTertiary Structure Of The Major House Dust Mite Allergen Der P 2, NMR, 10StructuresClassificationAllergenCompoundMol_Id: 1; Molecule: Mite Allergen Der P 2; Chain: Null; Engineered: Yes;Mutation: D1S; Other_Details:D1S Mutant Made To Enhance N-Terminal MetRemovalID NO: 1AHKDeposited: 07 April 1997 Exp. Method: NMR, Minimized Average StructureTitleDer F 2, The Major Mite Allergen From Dermatophagoides Farinae, NMR,Minimized Average StructureClassificationAllergenCompoundMol_Id: 1; Molecule: Der F 2; Chain: Null; Synonym: Der F II; Engineered: YesID NO: 1AHMDeposited: 07 April 1997 Exp. Method: NMR, 10 StructuresTitleDer F 2, The Major Mite Allergen From Dermatophagoides Farinae, NMR, 10StructuresClassificationAllergenCompoundMol_Id: 1; Molecule: Der F 2; Chain: Null; Synonym: Der F II; Engineered: YesID NO: 1B6FDeposited: 13 Jan. 1999 Exp. Method: NMR, 23 StructuresTitleBirch Pollen Allergen Bet V 1ClassificationPlant ProteinCompoundMol_Id: 1; Molecule: Major Pollen Allergen Bet V 1-A; Chain: A; Engineered:Yes; Mutation: YesID NO: 1BBGDeposited: 24 April 1998 Exp. Method: NMR, Minimized Average StructureTitleRagweed Pollen Allergen From Ambrosia Trifida V, NMR, Minimized AverageStructureClassificationAllergenCompoundMol_Id: 1; Molecule: Pollen Allergen 5; Chain: NullID NO: 1BJ7Deposited: 02 July 1998 Exp. Method: X-ray Diffraction Resolution: 1.80 ÅTitleBovine Lipocalin Allergen Bos D 2ClassificationAllergenCompoundMol_Id: 1; Molecule: D 2; Chain: Null; Synonym: Dander Major Allergen Bda20,Dermal Allergen Bda20; Engineered: Yes; Biological_Unit: MonomerID NO: 1BMWDeposited: 27 July 1998 Exp. Method: NMR, 38 StructuresTitleA Fibronectin Type III Fold In Plant Allergens: The Solution Structure Of Phl PiiFrom Timothy Grass Pollen, NMR, 38 StructuresClassificationAllergenCompoundMol_Id: 1; Molecule: Pollen Allergen Phl P2; Chain: Null; Synonym: Phl P II;Engineered: Yes; Biological_Unit: MonomerID NO: 1BTVDeposited: 30 Jan. 1997 Exp. Method: NMR, 20 StructuresTitleStructure Of Bet V 1, NMR, 20 StructuresClassificationMajor Birch Pollen AllergenCompoundMol_Id: 1; Molecule: Bet V 1; Chain: Null; Engineered: YesID NO: 1BV1Deposited: 08 July 1997 Exp. Method: X-ray Diffraction Resolution: 2.00 ÅTitleBirch Pollen Allergen Bet V 1ClassificationAllergenCompoundMol_Id: 1; Molecule: Bet V 1; Chain: Null; Synonym: Major Pollen Allergen BetV 1-A; Engineered: YesID NO: 1BWHDeposited: 24-Sep-1998 Exp. Method: X-ray Diffraction Resolution: 1.80 ÅTitleThe 1.8 A Structure Of Ground Control Grown Tetragonal Hen Egg WhiteLysozymeClassificationHydrolaseCompoundMol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: Gal D IV, Allergen Gal D4; Ec: 3.2.1.17ID NO: 1BWIDeposited: 24-Sep-1998 Exp. Method: X-ray Diffraction Resolution: 1.80 ÅTitleThe 1.8 A Structure Of Microbatch Oil Drop Grown Tetragonal Hen Egg WhiteLysozymeClassificationHydrolaseCompoundMol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: Gal D IV, Allergen Gal D4; Ec: 3.2.1.17ID NO: 1BWJDeposited: 18-Sep-1998 Exp. Method: X-ray Diffraction Resolution: 1.80 ÅTitleThe 1.8 A Structure Of Microgravity Grown Tetragonal Hen Egg WhiteLysozymeClassificationHydrolaseCompoundMol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: Gal D IV, Allergen Gal D4; Ec: 3.2.1.17ID NO: 1CQADeposited: 26 July 1996 Exp. Method: X-ray Diffraction Resolution: 2.40 ÅTitleBirch Pollen ProfilinClassificationContractile ProteinCompoundMol_Id: 1; Molecule: Profilin; Chain: Null; Engineered: YesID NO: 1E09Deposited: 15 March 2000 Exp. Method: NMR, 22 StructuresTitleSolution Structure Of The Major Cherry Allergen Pru Av 1ClassificationAllergenCompoundMol_Id: 1; Molecule: Pm Av 1; Chain: A; Engineered: YesID NO: 1EW3Deposited: 21 April 2000 Exp. Method: X-ray Diffraction Resolution: 2.30 ÅTitleCrystal Structure Of The Major Horse Allergen Equ C 1ClassificationAllergenCompoundMol_Id: 1; Molecule: Allergen Equ C 1; Chain: A; Engineered: YesID NO: 1F2KDeposited: 26 May 2000 Exp. Method: X-ray Diffraction Resolution: 2.30 ÅTitleCrystal Structure Of Acanthamoeba Castellanii Profilin II, Cubic Crystal FormClassificationStructural ProteinCompoundMol_Id: 1; Molecule: Profilin II; Chain: A, B; Engineered: YesID NO: 1FCQDeposited: 19 July 2000 Exp. Method: X-ray Diffraction Resolution: 1.60 ÅTitleCrystal Structure (Monoclinic) Of Bee Venom HyaluronidaseClassificationHydrolaseCompoundMol_Id: 1; Molecule: Hyaluronoglucosaminidase; Chain: A; Synonym:Hyaluronidase, Api M II; Ec: 3.2.1.35; Engineered: YesID NO: 1FCUDeposited: 19 July 2000 Exp. Method: X-ray Diffraction Resolution: 2.10 ÅTitleCrystal Structure (Trigonal) Of Bee Venom HyaluronidaseClassificationHydrolaseCompoundMol_Id: 1; Molecule: Hyaluronoglucosaminidase; Chain: A; Synonym:Hyaluronidase, Api M II; Ec: 3.2.1.35; Engineered: YesID NO: 1FCVDeposited: 19 July 2000 Exp. Method: X-ray Diffraction Resolution: 2.65 ÅTitleCrystal Structure Of Bee Venom Hyaluronidase In Complex With HyaluronicAcid TetramerClassificationHydrolaseCompoundMol_Id: 1; Molecule: Hyaluronoglucosaminidase; Chain: A; Synonym:Hyaluronidase, Api M II; Ec: 3.2.1.35; Engineered: YesID NO: 1FLQDeposited: 15 Aug. 2000 Exp. Method: X-ray Diffraction Resolution: 1.80 ÅTitleHen Egg White Lysozyme Mutant With Alanine Substituted For GlycineClassificationHydrolaseCompoundMol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: 1,4-N-AcetylmuramidaseC, Allergen Gal D 4, Gal D IV; Ec: 3.2.1.17; Engineered: Yes; Mutation: YesID NO: 1FLUDeposited: 15 Aug. 2000 Exp. Method: X-ray Diffraction Resolution: 1.78 ÅTitleHen Egg White Lysozyme Mutant With Alanine Substituted For GlycineClassificationHydrolaseCompoundMol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: 1,4-N-AcetylmuramidaseC, Allergen Gal D 4, Gal D IV; Ec: 3.2.1.17; Engineered: Yes; Mutation: YesID NO: 1FLWDeposited: 15 Aug. 2000 Exp. Method: X-ray Diffraction Resolution: 1.81 ÅTitleHen Egg White Lysozyme Mutant With Alanine Substituted For GlycineClassificationHydrolaseCompoundMol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: 1,4-N-AcetylmuramidaseC, Allergen Gal D 4, Gal D IV; Ec: 3.2.1.17; Engineered: Yes; Mutation: YesID NO: 1FLYDeposited: 15 Aug. 2000 Exp. Method: X-ray Diffraction Resolution: 1.83 ÅTitleHen Egg White Lysozyme Mutant With Alanine Substituted For GlycineClassificationHydrolaseCompoundMol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: 1,4-N-AcetylmuramidaseC, Allergen Gal D 4, Gal D IV; Ec: 3.2.1.17; Engineered: Yes; Mutation: YesID NO: 1FN5Deposited: 21 Aug. 2000 Exp. Method: X-ray Diffraction Resolution: 1.78 ÅTitleHen Egg White Lysozyme Mutant With Alanine Substituted For GlycineClassificationHydrolaseCompoundMol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: 1,4-N-AcetylmuramidaseC, Allergen Gal D 4, Gal D IV; Ec: 3.2.1.17; Engineered: Yes; Mutation: YesID NO: 1FSKDeposited: 11 Sept. 2000 Exp. Method: X-ray Diffraction Resolution: 2.90 ÅTitleComplex Formation Between A Fab Fragment Of A Monoclonal IgG Antibodyand The Major Allergen From Birch Pollen Bet V 1ClassificationImmune SystemCompoundMol_Id: 1; Molecule: Major Pollen Allergen Bet V 1-A; Chain: A, D, G, J;Synonym: Bet V I-A, Betvi Allergen; Engineered: Yes Mol_Id: 2; Molecule:Immunoglobulin Light Chain; Chain: B, E, H, K; Synonym: Bv16 Fab-Fragment,Mopc21 Coding Sequence; Engineered: Yes Mol_Id: 3; Molecule: AntibodyHeavy Chain Fab; Chain: C, F, I, L; Synonym: Heavy Chain Of The MonoclonalAntibody Mst2; Engineered: YesID NO: 1G5UDeposited: 02 Nov. 2000 Exp. Method: X-ray Diffraction Resolution: 3.10 ÅTitleLatex Profilin Hevb8ClassificationAllergenCompoundMol_Id: 1; Molecule: Profilin; Chain: A, B; Engineered: YesID NO: 1H6MDeposited: 19 June 2001 Exp. Method: X-ray Diffraction Resolution: 1.64 ÅTitleCovalent Glycosyl-Enzyme Intermediate Of Hen Egg White LysozymeClassificationHydrolase (O-Glycosyl)CompoundMol_Id: 1; Molecule: Lysozyme C; Synonym: 1,4-N-Acetylmuramidase C,Allergen Gal D 4, Gal D IV; Chain: A; Ec: 3.2.1.17; Engineered: Yes; Mutation:Yes; Other_Details: Covalent 2-Fluorochitobiosyl Enzyme IntermediateID NO: 1JTIDeposited: 21 Aug. 2001 Exp. Method: X-ray Diffraction Resolution: 2.30 ÅTitleLoop-Inserted Structure OfP1-P1′ Cleaved Ovalbumin Mutant R339TClassificationAllergenCompoundMol_Id: 1; Molecule: Ovalbumin; Chain: A, B; Engineered: Yes; Mutation: YesID NO: 1JTTDeposited: 22 Aug. 2001 Exp. Method: X-ray Diffraction Resolution: 2.10 ÅTitleDegenerate Interfaces In Antigen-Antibody ComplexesClassificationImmune System, LysozymeCompoundMol_Id: 1; Molecule: Vh Single-Domain Antibody; Chain: A; Fragment: VhDomain Fragment; Engineered: Yes Mol_Id: 2; Molecule: Lysozyme; Chain: L;Fragment: Enzyme; Synonym: 1,4-N-Acetylmuramidase C, Allergen Gal D IV;Ec: 3.2.1.17ID NO: 1K0KDeposited: 19 Sept. 2001 Exp. Method: X-ray Diffraction Resolution: 2.35 ÅTitleYeast Profilin, Cubic Crystal FormClassificationContractile ProteinCompoundMol_Id: 1; Molecule: Profilin; Chain: A; Engineered: YesID NO: 1KKCDeposited: 07 Dec. 2001 Exp. Method: X-ray Diffraction Resolution: 2.00 ÅTitleCrystal Structure Of Aspergillus Fumigatus MnsodClassificationOxidoreductaseCompoundMol_Id: 1; Molecule: Manganese Superoxide Dismutase; Chain: A, B, X, Y;Synonym: Mnsod; Ec: 1.15.1.1; Engineered: YesID NO: 1KURDeposited: 22 Jan. 2002 Exp. Method: Theoretical ModelTitleTheoretical Model Of The Allergen Jun A 3 From Mountain Cedar PollenClassificationAllergenCompoundMol_Id: 1; Molecule: Allergen Jun A 3; Chain: A; Synonym:Pathogenesis-Related ProteinID NO: 1PLMDeposited: 09 Jan. 1998 Exp. Method: Theoretical ModelTitleArabidopsis Profilin 1 Complexed With Poly-L-Proline, Theoretical ModelClassificationComplex (Protein/Peptide)CompoundMol_Id: 1; Molecule: Profilin 1; Chain: A; Engineered: Yes Mol_Id: 2; Molecule:Poly-L-Proline; Chain: B; Engineered: YesID NO: 1PRQDeposited: 18 Aug. 1997 Exp. Method: X-ray Diffraction Resolution: 2.50 ÅTitleAcanthamoeba Castellanii Profilin IaClassificationContractile ProteinCompoundMol_Id: 1; Molecule: Profilin Ia; Chain: Null; Engineered: YesID NO: 1QMRDeposited: 06 Oct. 1999 Exp. Method: X-ray Diffraction Resolution: 2.15 ÅTitleBirch Pollen Allergen Bet V 1 Mutant N28T, K32Q, E45S, P108GClassificationAllergenCompoundMol_Id: 1; Molecule: Major Pollen Allergen Bet V 1-A; Chain: A; Synonym: BetV 1; Engineered: Yes; Mutation: YesID NO: 1QNXDeposited: 25 Oct. 1999 Exp. Method: X-ray Diffraction Resolution: 1.90 ÅTitleVes V 5, An Allergen From Vespula Vulgaris VenomClassificationAllergenCompoundMol_Id: 1; Molecule: Ves V 5; Chain: A; Synonym: Antigen 5; Engineered: YesID NO: 1WHODeposited: 04 April 1997 Exp. Method: X-ray Diffraction Resolution: 1.90 ÅTitleAllergen Phl P 2ClassificationAllergenCompoundMol_Id: 1; Molecule: Allergen Phl P 2; Chain: Null; Synonym: Phl P II;Engineered: YesID NO: 1WHPDeposited: 04 April 1997 Exp. Method: X-ray Diffraction Resolution: 3.00 ÅTitleAllergen Phl P 2ClassificationAllergenCompoundMol_Id: 1; Molecule: Allergen Phl P 2; Chain: Null; Synonym: Phl P II;Engineered: YesID NO: 2BBGDeposited: 24 April 1998 Exp. Method: NMR, 30 StructuresTitleRagweed Pollen Allergen From Ambrosia Trifida V, NMR, 30 StructuresClassificationAllergenCompoundMol_Id: 1; Molecule: Pollen Allergen 5; Chain: NullID NO: 3BBGDeposited: 24 April 1998 Exp. Method: NMR, 2 StructuresTitleMulti-Conformer Structure Of Ragweed Pollen Allergen From Ambrosia TrifidaV, NMR, 2 StructuresClassificationAllergenCompoundMol_Id: 1; Molecule: Pollen Allergen 5; Chain: NullID NO: 3NULDeposited: 27 Nov. 1996 Exp. Method: X-ray Diffraction Resolution: 1.60 ÅTitleProfilin I From Arabidopsis ThalianaClassificationActin-Binding ProteinCompoundMol_Id: 1; Molecule: Profilin I; Chain: Null; Engineered: SelenomethionylProtein

[0447]

Recombinant hybrid allergen constructs with reduced allergenicity that retain immunogenicity of the natural allergen

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