AU2002245597A1

AU2002245597A1 - Recombinant hybrid allergen constructs with reduced allergenicity that retain immunogenicity of the natural allergen

Info

Publication number: AU2002245597A1
Application number: AU2002245597A
Authority: AU
Inventors: Te Piao King; Michael Dho Spangfort
Original assignee: ALK Abello AS; Rockefeller University
Current assignee: ALK Abello AS; Rockefeller University
Priority date: 2001-03-02
Filing date: 2002-03-04
Publication date: 2002-09-19

Description

RECOMBINANT HYBRID ALLERGEN CONSTRUCTS

WITH REDUCED ALLERGENICITY THAT RETAIN

IMMUNOGENICITY OF THE NATURAL ALLERGEN

This application claims priority under 35 U.S.C. § 119 (e) of U.S. Provisional

Application Serial No. 60/272,818, filed March 2, 2001, which is hereby incorporated herein by reference in its entirety.

FIELD OF THE INVENTION 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

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.

Biochemical Aspects of Allergens 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). 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. Immunol. 79:113; Hoffman, 1985, J. Allergy and Clin. 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. 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 (Rafhar 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. T and B Cell Epitope of Allergens

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 polymoφhic 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.

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 et al, 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: Thl 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).

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).

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; OΗehir et al, 1991, Ann. Rev. Immunol. 9:67).

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.

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. OΗehir et al, supra). These allergens include ragweed Amb HI, rye grass Lol p I, cat Fel d I, mouse urine Mus m I, midge Chi 11, 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. Modulation of T and B Cell Responses

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.

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. 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 autoimmime response (Gaur et al, 1992, Science 259:1491-1494).

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. Immunotherapy

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.)

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. OΗehir et al, 1991, supra). 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).

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). 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.

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 Thl 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.

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. 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

-1 - 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 (OΗehir et al, 1991, J. Allergy Clin. Immunol. 87:1120). 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?

U.S. Patent Nos. 5,593,877; 5,612,209, 5,804,201, 6,106,844, 6,270,763 and 6,287,559 and U.S. application Serial 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.

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. 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.

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.

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. 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

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.

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.

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.

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.

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.

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

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.

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 5 peptide selected from the group consisting of:

NNYCKIKC (SEQ ID: 1);

NNYCKIKCLKGGNHTACK (SEQ ID: 2); NNYCKIKCLKGGNHTACKYGSLKP (SEQ ID: 3);

ΝΝYCKIKCLKGGVHTACKYGSLKPΝCGΝKVW(SEQID: 4);

ΝΝYCKTKCLKGGNHTACKYGSLKPΝCGΝKVVNSYGLTKQ (SEQ ID:

5);

ΝΝYCKD CLKGGVHTACKYGSLKPΝCGΝKNvNSYGLTKQEKQDILK (SEQ ID: 6);

QNGQΝVALTGSTAAKYDDPVKLλ^KMWEDEVKDYΝPKKKFSGΝDFL

KTG (SEQ ID NO: 7); HYTQMVWANTKEVGCGSIKYIQEKWHKHYLNCNYGPSGNFKNEELY QTK (SEQ ID NO: 8) LKPNCGNKNW (SEQ ID NO: 9);

LTGSTAAKYDD (SEQ ID NO: 10); PKKKFSGND (SEQ ID NO: 11) IQEKWHK (SEQ ID NO: 12); and FKNEELYQTK (SEQ ID NO: 13); NNYCKIKCLKGG\ΗTACKYGSLKPNCGNKNVNSYGLTKQEKQD1LK

EHΝD (SEQ ID ΝO: 93); NNYCKIKCLKGGVHTACKYGSLKPNCGNKNNVSYGLTKQEKQDrLK

EHΝDFRQKIAR (SEQ ID NO: 94); NNYCKTKCLKGGNHTACKYGSLKPNCGNKNNNSYGLTKQEKQDILK EHΝDFRQKIARGLETRGΝPGPQPPAKΝMKΝ (SEQ ID NO: 95).

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 hCMN, 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 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.

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.

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. 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.

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.

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.

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.

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.

ABBREVIATIONS Dol m Dolichovespula maculata white faced hornet Dol a D. arenaria yellow hornet

Pol a Polistes annularis wasp Pol e P. exclamans wasp

Ves m Vespula maculifrons yellowjacket Ves v V. vulgaris yellowjacket

PCR polymerase chain reaction

RACE rapid amplification of cDNA ends

TCR T cell receptor for antigen

BRIEF DESCRIPTION OF THE DRAWINGS

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

Fig. 3. Amino acid comparison of Ves v 5 (V) [SEQ ID NO: 16] and Pol a 5 (P) [SEQ ID NO: 17]. Fig. 4. Schematic sequence representations of Ag 5s and hybrids. Residue numbers given for hybrids refer to those of Ves v 5.

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

Fig. 6A-B. SDS gel patterns of Ag 5s and hybrids.

Fig. 7. Circular dichroism (CD) spectra of Ves v 5 and hybrids. 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.

Fig. 9A-C. Inhibition ELISA with sera from yellow jacket-sensitive patients. Fig. 10 A-C. Binding of mouse Ves v 5-specific monoclonal antibodies to solid-phase Ves v 5 or hybrids.

Fig. 11 A-C. Histamine release assay of Ves v 5, Pol a 5 and hybrids.

Fig. 12A-B. Alignment of Ves v 5-like proteins. Aligned proteins are Ves v 5 [SEQ ED NO: 81]; Sol i 3 [SEQ ED NO: 82]; Lycopersicon esculentum pl4a [SEQ ED NO: 83]; Schizophyllum commune SC7 [SEQ ED NO: 84]; human trypsin inhibitor [SEQ ED NO: 85]; human glipr [SEQ ED NO: 86]; Heloderma horridum helothermine [SEQ ED NO: 87]; and human TPX-1 [SEQ ED NO: 88].

DETAILED DESCRIPTION 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 comer 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. 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.

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.

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

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.

Clinical studies in patients and tests with experimental animals have shown that there is limited cross reactivity of antibodies specific for the yellowjacket 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 yellowjacket 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).

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. 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.

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.

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. 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.

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.

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.

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.

En 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. 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.

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%.

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 A) 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 Pinoles including birch (Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive (Olea), the order oϊPoales including i.a. grasses of the genera Lolium, Phleum, Poa, Cynodon, Dactylis and Secale, the orders ol Aster ales and Urticales including herbs of the genera Ambrosia 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 orbiting 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), Aln g 1 (Alnus glutinosa, alder), Cora 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, Dae g 1, Fes p l, Hol l , Lolp 1 and 5, Pas n \, Phlp 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), Derfl and 2, Derp 1 and 2 (house dust mites, D.fαrinαe andEA pteronyssinus, respectively), Lep d 1 and 2 (Lepidoglyphus destructor; storage mite), Blα g 1 and 2, Eer α 1 (cockroaches, Blαtellα germanica and Periplaneta americana, respectively), Fel d 1 (cat), Canfl (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. 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 IgΕ type antibodies. The IgΕ type antibodies are responsible for mediating the symptoms of an allergic condition, i.e., immediate-type hypersensitivity. 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).

The taxonomic classification of Vespula vulgaris is as follows:

Order: Hymenoptera

Suborder: Apocrita

Division: Aculeata

Superfamily: Vespoidea

Family: Vespidae

Subfamily: Vespinae

Genus: Vespula

Species Group: Vespula vulgaris species group

Species: vulgaris

The taxonomic classification for Polistes annul.'

Order: Hymenoptera

Suborder: Apocrita

Division: Aculeata

Superfamily: Vespoidea

Family: Vespidae

Subfamily: Polistinae

Tribe: Polistini

Genus: Polistes

Subgenus: Aphanilopterus

Species: annularis

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.

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., HEV, Herpes Simplex virus, or papilloma virus; an autoimmune disease, e.g., arthritis or Lupus.

Determining Allergen Structure 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 A 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 A 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. 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, WE). 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. 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. 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 absoφtion 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. Molecular Biological Techniques 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, New York (herein "Sambrook et al, 1989"); "DNA Cloning: a Practical Approach," Volumes I and EI (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & SJ.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" [ERL 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. Patent Nos. 5,593,877; 5,612,209, 5,804,201, 6,106,844 and U.S. application Serial Nos. 08/484,388, 08/474;853, and 09/166,205 to King and in Monsalve et al. (1999, Protein Expr. Purif. 16:410).

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.

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., 5x SSC, 0.1% SDS, 0.25% nonfat dry milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5x or 6x SSC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5x or 6x 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.

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. 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.

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.

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 puφoses 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 SI), 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.

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.

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. 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., 5x SSC, 0.1% SDS, 0.25% nonfat dry milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher Tm, e.g., 40% formamide, with 5x or 6x SSC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5x or 6x 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. 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 prefened embodiment, the Tm is 65 °C.

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.

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. 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 puφoses 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 SI), 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. A coding sequence is "under the controF'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.

Nucleic Acid Molecules Encoding Hybrid Proteins The invention relates to isolated nucleic acid molecules encoding recombinant allergen hybrid proteins. The invention further relates to a cell line stably containing a recombinanfnucleic 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. 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 ED NO: 14; see Fig. 1) and Pol a Ag 5 (SEQ ED NO: 15; see Fig. 2). Also provided are the amino acid sequences of Ves v Ag 5 (SEQ ED NO: 16; see Fig. 1) and Pol a Ag5 (SEQ ED NO: 17; see Fig. 2). En 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™).

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.

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.

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

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.

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, New York). 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.

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 prefened 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). 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 prefened 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 Coφ., San Diego, CA). 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.

In specific embodiments, transformation of host cells with recombinant DNA molecules that incoφorate 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.

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

Expression of an Allergen Hybrid Protein

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 maybe used. 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). 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. 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. 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, CA). 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.

En another embodiment, a periplasmic foπn 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. 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/trarislational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination).

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 heφes 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 j8-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. 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.

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 March 15, 1990).

Both cDNA and genomic sequences can be cloned and expressed. 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.

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. 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 prefened aspect, the proteins are further purified by reverse phase chromatography.

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

En 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.

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 ED NO: 1-13 or 93-95. 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 ED NO: 17.

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 maybe 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. 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.

In a particular embodiment, the hybrid protein is expressed in an insect cell expression system, e.g., using a baculovirus expression vector. In a prefened 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.

Activity Assays With Hybrid Proteins of the Invention 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 conect 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.

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 .

In a prefened 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 incoφoration of ³H-thymidine, which increases with DNA synthesis associated with proliferation.

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.

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 etal, 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, BioTechmques 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., EL-4 and EL-5 (Purkeson and Isakson, 1992, J. Exp. Med. 175:973).

Thus, in a prefened 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. , EL-4. Such assays may indicate which component or components of the hybrid protein are responsible for the allergic condition.

Therapeutic Uses of the Hybrid Protein and Nucleic Acid Vectors

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.

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.

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.

Therapeutic Methods

Therapeutic compositions of the invention (see, infra) can be used in immunotherapy, also refened 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.

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.

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).

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 etal, 1993, J. Exp. Med. 178:1783-88). 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.

These results can also be achieved by administration of a vector that permits expression of the hybrid protein, . e. , by gene therapy. Prefened vectors, particularly for cellular assays in vitro and in vivo, are viral vectors, such as lentiviruses, retroviruses, heφes viruses, adenovimses, adeno-associated viruses, vaccinia viras, baculovims, alphavimses (especially Sindbis viruses and Semliki Forest virases), 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 prefened, 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. 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.

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, BioTechmques 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 vims is a minimal vims, i.e., it retains only the sequences of its genome that are necessary for encapsidating the genome to produce viral particles.

DNA viral vectors include an attenuated or defective DNA vims, such as but not limited to, heφes simplex viras (HSV), papillomavirus, Epstein Ban vims (EBV), adenovims, adeno-associated viras (AAV), alphavims (especially Sindbis vims), and the like. Defective viruses that entirely or almost entirely lack viral genes are prefened. Defective vims is not infective after introduction into a cell. Use of defective viral vectors allows for admimstration 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 heφes virus 1 (HSV1) vector (Kaplitt et al, Molec. Cell. Neurosci. 1991, 2:320-330), defective heφes viras vector lacking a glyco-protein L gene, or other defective heφes viras 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 viras and Semliki Forest virus-based vectors (U.S. Patent No. 5,091,309; PCT Publication No. WO 98/44132; Schlesinger and Dubensky, Cun. Opin. Biotechnol. 1999, 5:434-9; Zaks et al., Nat. Med. 1999, 7:823-7). Various companies produce viral vectors commercially, including, but not limited to, Avigen, Inc. (Alameda, CA; AAV vectors), Cell Genesys (Foster City, CA; retroviral, adenoviral, AAV, and lentiviral vectors), Clontech (retroviral and baculoviral vectors), Genovo, Inc. (Sharon Hill, PA; adenoviral and AAV vectors), Genvec (France; adenoviral vectors), EntroGene (Leiden, Netherlands; adenoviral vectors), Molecular Medicine (retroviral, adenoviral, AAV, and heφes viral vectors), Norgen (adenoviral vectors), Oxford BioMedica (Oxford, United Kingdom; lentiviral vectors), and Transgene (Strasbourg, France; adenoviral, vaccinia, retroviral, and lentiviral vectors).

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. Patent No. 5,459,127. Lipids may be chemically coupled to other molecules for the puφose 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.

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). 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. Patent 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, CP. Acad. Sci. 1988, 321:893; PCT Publication Nos. WO 99/01157, WO 99/01158, and WO 99/01175). Treatment of Immune System Related Diseases

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 virases including, but not limited to, HEV, Heφes Simplex viras, 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. HEV, Heφes Simplex viras, or papilloma vims; or an autoimmune disease, e.g. arthritis or Lupus. 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 HIN, 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. Pharmaceutically Acceptable Compositions 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. 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 prefened 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.

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.

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

Primers 1-24 used in the Examples are listed in Table 1. Ves v 5 EA and KR constracts were prepared by PCR amplification of Ves v 5 cDNA template (Lu et al, 1993, J. Immunol. 150:2823) with the primers 1 (SEQ ED NO: 31) and 3 (SEQ ED NO: 33) or 2 (SEQ ED NO: 32) and 3 (SEQ ED 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 ED NO: 34) and 6 (SEQ ED NO: 24) or 5 (SEQ ED NO: 35) and 6 (SEQ ED NO: 36), respectively. Each cDNA construct contained an EcoRI or Xhol site at the 5 'terminus and an Xbal site at the 3 '-terminus. cDNAs were cloned in the plasmid vector pPICZαA (Invitrogen Coφ, San Diego, CA) as either EcoRI -Xbal or Xhol-Xbal 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 constracts were prepared as described in King et al. (2001, J. Immunol. 166:6057-6065).

(i) PV1-46. The PV1-46 hybrid was constructed by joining ammo-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 conesponds to a Bsi HKA I restriction enzyme cleavage site.

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 ED NO: 34) and 8 (SEQ ED 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 ED NO: 37) and 6 (SEQ ED 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 ED NO: 34) and 6 (SEQ ED NO: 36), yielding a full length Pol a 5 with the EH-encoding sequence converted to a Bsi HKA I site.

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.

(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 ED NO: 39) and 10 (SEQ ED NO: 40). En 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 ED NO: 41) and 12 (SEQ ED NO: 42). Hybrid PV109-155 encoding cDNA was prepared by ligation of the appropriate fragments from Apo 1 digestions of converted Pol a 5 and converted P V 1 - 155 encoding cDNAs into pPICZαA.

(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. (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 ED NO: 32) and 6 (SEQ ED NO: 36). PV1-18 was prepared using primers 6 (SEQ ED NO: 36) and 13 (SEQ ED NO: 43). PV195-204 was prepared using primers 4 (SEQ ED NO: 34) and 14 (SEQ ED NO: 44). The hybrids were cloned into pPICZαA. (v) PV1-24, PVI-32 , PV1-39, PV1-50, PVI-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 ED NO: 31) and 15 (SEQ ED NO: 45) with Ves v 5 cDNA as template and primers 6 (SEQ ED NO: 36) and 16 (SEQ ED 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 ED NO: 31) and 6 (SEQ ED NO: 36) to yield PV1-24. For PVI-32, first round PCRs were conducted using primers 1 (SEQ ED NO: 31) and 18 (SEQ ED NO: 48) with Ves v 5 cDNA as template and primers 6 (SEQ ED NO: 36) and 17 (SEQ ED 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 ED NO: 31) and 6 (SEQ ED NO: 36) to yield PV1-24. For PV1-39, first round PCRs were conducted using primers 2 (SEQ ED NO: 32) and 19 (SEQ ED NO: 49) with Ves v 5 cDNA as template and primers 6 (SEQ ED 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 ED NO: 36) to yield PV1-39. For PV1-50, first round PCRs were conducted using primers 2 (SEQ ED NO: 32) and 28 (SEQ ED NO: 58) with Ves v 5 cDNA as template and primers 6 (SEQ ED NO: 36) and 27 (SEQ ED 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 ED NO: 32) and 6 (SEQ ED NO: 36) to yield PV1-50. For PV1-57, first round PCRs were conducted using primers 2 (SEQ ED NO: 32) and 30 (SEQ ID NO: 60) with Ves v 5 cDNA as template and primers 6 (SEQ ED 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 ED NO: 32) and 6 (SEQ ED NO: 36) to yield PV1-57. For PV1-76, first round PCRs were conducted using primers 2 (SEQ ED NO: 32) and 32 (SEQ ED NO: 62) with Ves v 5 cDNA as template and primers 6 (SEQ ED NO: 36) and 31 (SEQ ED 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 ED NO: 32) and 6 (SEQ ED NO: 36) to yield PV1-76. Hybrid cDNAs were cloned into pPICZαA.

(vi) PV22-32, PV115-125, PV142-150 andPV176-182. These constracts are hybrid Ag 5s wherein short Ves v 5 polypeptides replace homologous sequences in otherwise intact full length Pol a 5.

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.

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

Table 1. Primers for preparation of Ves v and Pol a 5s and their hybrids.

Primer Sequence (5' to 3')

1 CGTGAATTCAACAATTATTGTAAAATAAAA (SEQ ID NO: 31) 2 CGTCTCGAGAAAAGAAACAATTATTGTAAAATAAAA (SEQ ID N0: 32)

3 CGTTCTAGATTACTTTGTTTGATAAAGTTC (SEQ ID NO: 33)

4 CGTGAATTCGTTGATTATTGTAAAATAAAA (SEQ ID NO: 34)

5 CGTCTCGAGAAAAGAGTTGATTATTGTAAAATAAAA (SEQ ID NO: 35)

6 CGTTCTAGATTATTπTTTGTATAAGGTAG (SEQ ID NO: 36) 7 GTAAGCGAGCACAATCGGTTT (SEQ ID NO: 37)

8 AAACCGATTGTGCTCGCTTAC (SEQ ID NO: 38)

9 GTAGCAAAATTTCAGGTTGGA (SEQ ID NO: 39)

10 TCCAACCTGAAATTTTGCTAC (SEQ ID NO: 40)

11 ACCGCAAAATTTCCAGTTGGA (SEQ ID NO: 41) 12 TCCAACTGGAAATTTTGCGGT (SEQ ID NO: 42)

13 CGTGAATTCAACAATTATTGTAAAATAAAATGTTTGAAAGGAGGTGTCCATACTGCCT GCAAATATGGAGAA (SEQ ID NO: 43) 14 CGTΓCTAGATTACTTΓGTTΓGATAAAGT CCTCATTCTΓAAAATT CCAGCTGG (SEQ ID

NO: 44)

15 GGCACAATTCTTGCTCGGTTTAAGACTTCCATA (SEQ ID NO: 45)

16 TATGGAAGTCTTAAACCGAGCAAGAATTGTGCC (SEQ ID NO: 46)

17 CTTAAACCGAATTGCGGTAATAAGGTAGTGGTATCGGTTGGTCCA (SEQ ID NO: 47)

18 TGGACCAACCGATACCACTACCTTATTACCGCAATTCGGTTTAAG (SEQ ID NO: 48)

19 TATGGTCTAACGAAACAAGAGAAAAAATTAATCGTA (SEC ID NO: 49)

20 TACGAT^AATTTTT CTCTTGTTTCGTTAGACCATA (SEC ID NO: 50)

21 TTAACAGGTAGCACGGCTGCTAAATACGATGATGTAGTCAGTCTA (SEQ ID NO: 51)

22 ATCATCGTATTTAGCAGCCGTGCTACCTGTTAACGCTATATTTTG (SEQ ID NO: 52) 23 CCTAAGAAAAAGTTTΓCGGGAAACGACTTTGCTAAAATTGGC (SEQ ID NO: 53)

24 GTCGTTΓCCCGAAAACTTTTTCTTAGGATTAAAATCTTTCAC (SEQ ID NO: 54)

25 ATTCAAGAGAAATGGCACAAACATTACCTCATA (SEQ ID NO: 55)

26 TTTGTGCCATTTCTCTTGAATATATTTTAGAGA (SEQ ID NO: 56)

27 GAGCACAATGACTTTAGACAAAAA (SEQ ID NO: 57)

28 TTTTTGTCTAAAGTCATTGTGCTC (SEQ ID NO: 58)

29 AAAATTGCACGAGGGTTGGAAACA (SEQ ID NO: 59) 30 TGTT CCAACCCTCGTGCAATTTT (SEQ ID NO: 60)

31 AATATGAAAAATTTGGTATGGAAC (SEQ ID NO: 61)

32 GTTCCATACCAAATTTTTCATATT (SEQ ID NO: 62)

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, CA). 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 conesponded 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 polymoφhism, 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 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 x 10⁹ cells in 40 μl of IM sorbitol) by electroporation. Transformed cells were diluted to 2 ml with IM sorbitol and allowed to recover at 30° for 1 hr without shaking and for an additional hour with shaking at 200 φm. 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 1/30 scale and the culture fluids were screened by SDS gel electrophoresis for secreted proteins.

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 φm to an A^on_m 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 φm for 4-6 days with daily addition of 1 ml of 50% methanol.

Ag 5 s 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 C 18 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. 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 1mm path length in an AVIV 62DS spectrometer.

Example 3 Physico-chemical characterization of recombinant vespid

Ag 5s and hybrids

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).

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).

Recombinant Ag 5 s 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 El (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).

The recombinant Ag 5 s 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. Results of mass spectrometric analysis of Ag 5 s 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).

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 v5, Pol a 5, and hybrids KR-PVl-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.

Table 2. Mass spectrometric data of recombinant vespid Ag 5s and hybrids.

Mass units

Protein Assumed sequence Abundance¹ calc'd found

EA-Ves v 5 EAEAEF-Vv 80% 23,954 23,947

EAEF-Vv 20% 23,754 23,752

EA-Pol a 5 EAEAEF-Pa 100% 23,611 23,613

EA-PV1-18 EAEF-PV 43% 23,497 23,506

EAEAEF-PV 36% 23,697 23,698

REAEAEF-PV 21% 23,871 23,827

EA-PV1-18 EAEAEF-PV 100% 23,697 23,701

EA-PV1-32 EF-PV 60% 22,964 22,930

EAEF-PV 40% 23,151 23,134

EA-PV1-46 EAEF-PV 53% 23,300 23,327

EAEAEF-PV 47% 23,500 23,515

EA-PV1-46 EF-PV 10% 23,099 23,109

EAEF-PV 50% 23,300 23,327

EAEAEF-PV 40% 23,500 23,515

EA-PV1-155 EF-PV 53% 23,375 23,334

EAEF-PV 47% 23,575 23,533

EA-PV22-32 EAEF-PV 55% 23,135 23,203

EAEAEF-PV 45% 23,336 23,371

EA-PV115-125 EAEAEF-PV 100% 23,873 23,887

EA-PV142-150 EAEAEF-PV 100% 23,592 23,585

EA-PV156-204 EAEF-PV 59% 23,776 23,775

EAEAEF-PV 41% 23,932 23,939

EA-PV195-204 EAEAEF-PV 70% 23,700 23,688

REAEAEF-PV 30% 23,874 23,844 KR-Ves v 5 Vv5 90% 23,277 23,274

EEGVSLEKR-Vv 10% 24,305 24,298

KR-Ves v 5 Vv 95% 23,277 23,284

EEGVSLEKR-Vv 5% 24,305 24,300

KR-Pol a 5 Pa 20% 22,934 22,951

EEGVSLEKR-Pa 80% 23,962 23,992

KR-Pol a 5 Pa 10% 22,934 22,935

EEGVSLEKR-Pa 90% 23,962 23,962

KR-PVl-24 PV 85% 22,903 22,897

EEGVSLEKR-PV 15% 23,931 23,933

KR-PV1-46 PV 70% 22,823 22,834

KR-PV 30% 23,107 23,157

KR-PV1-46 PV 60% 22,823 22,834

KR-PV 40% 23,107 23,157

'Protein 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.

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

ELISA was performed in 96-well plates in the wells coated with 4 μg/ml Ag 5 in 0.05 M Tris-HCl buffer of pH 8. Bound Igd was detected with 2 μg/ml biotinylated goat anti-mouse IgG (γl 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

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). 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 seram sample are shown in Fig. 8 A. At the highest concentration of 50 or 500μg/ml inhibitor tested, the two N-terminal hybrids EA-PVl-46 and EA-1-155 showed maximal inhibition approaching 100%, as did EA- or KR-Ves v 5. Two other N-terminal hybrids KR-PVl-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-PV 156-204 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. Ves v 5-specific B cell epitopes were also detected by inhibition analyses with sera from six yellowjacket sensitive patients. The data from three patients are shown in Fig. 9A-C. The results were similar to those obtained with mouse IgGs.

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.

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).

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 (Horaick and Karash, 1972, Immunochemistry 9:325; Crothers and Metzger, 1972, Irnmunochemistry 9:341). 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 5 (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-PVl-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-PVl-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-PV 1-32 and EA-PV 1-46 and moderate binding to EA-PV1-18 and EA-PV 1-24 (Fig. IOC). 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

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. 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 absoφtion 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 3 A. EA-PVl-46 gave a higher antibody response in set A mice than KR-PV 1-46 did in set B mice. This difference may be due to the different sets of mice used. EA-PV1-18 was used in both sets of experiments, and it gave higher antibody response in set A mice than that in set B mice.

Comparison of antibody levels in the N-terminal hybrid-specific sera samples in Table 3, before and after Pol a 5 absoφtion, 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 Nes v 5 than those on solid-phase hybrid suggest that the majority of hybrid- specific antibodies recognize overlapping regions of Nes v 5 and Pol a 5 in the hybrid. The data in set A of Table 3A indicated that of the three Ν-terminal hybrids, PN1-155 was as immunogenic as Nes v 5 was, PNI -46 was half as imunogenic as Nes v 5 and PVl-18 was about l/9th as immunogenic as Ves v 5. The data in set B indicate that PVl-46 and 1-32 were more immunogenic than PV1-24 and 1-18. The data from both sets suggest that the longer Ν-terminal hybrids PVl-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.

Table 3A

Murine antibody responses to vespid antigen 5s and hybrids mg/ml specific IgG in sera

Immunogen¹ by ELISA on solid-phase ^w

EA-PoI a 5

SET EA-Ves v 5 Hybrid

A KR-Ves v 5 8.9 (8.5) 0.6 -

KR-Pol a 5 2.8 (1.0) 7.0 -

EA-PV1-155 12.0 0.7 -

EA-PVl-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) 14.0 10.0 (0.3)

B KR-Ves v 5 15.0 (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-PVl-24 0.4 (0.3) 4.2 6.5 (0.9)

EA-PV1-18 0.4 (0.3) 4.5 5.3 (0.7)

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

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. 3. Values in parenthesis were obtained after absoφtion of V₅₀o diluted sera with 0.2 mg/ml EA-Pol a 5. The results shown in Table 3 A 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 IgGl content specific for Ves v, Pol a or hybrid, and percent of specific IgGl remaining after absoφtion with Pa.

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 <PVl-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 >PVl-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. Hybrids with Ves v contents of <PVl-32 show 2-4 mg/ml of Pol a-specific antibody, and hybrids with Ves v contents of >PVl-39 showed 0.04-1.34 mg/ml of Pol a-specific antibody. As the Ves v content of hybrids was increased from PVI-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.

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

Table 3B. Murine antibody responses to vespid antigen 5s and hybrids

Groups Ves v 5 specific Hybrid specific IgGl;

Construct of mice IgGl; % Ves v Pol a 5 specific IgGl % Ves v

Pol a 5 1 1.80 mg/ml; 64% 4.50 mg/ml

Ves v 5 4 10.7 ± 3.2 mg/ml 0.2 ±0.1 mg/ml 104 ± 15%

PV1-8 1 0 8.2 mg/ml

PVl-18 4 0.6 ±0.44 mg ml; 4.1 ±2.0 mg/ml 7.5 ±4.5 mg/ml; 68 ± 14% 27 ±26% PV1-24 2 0.35 ±0.06 mg/ml; 2.26 ±0.50 mg/ml 5.86 ±1.30 mg/ml; 81 ±17% 20 ±12%

PVI-32 3 0.52 ±0.39 mg/ml; 3.77 ±1.89 mg/ml 6.82 ±3.46 mg/ml; 34 ±25% 15 ±6%

PV1-39 2 4.45 ±0.70 mg/ml; 1.72 ±0.06 mg/ml 8.25 ±1/87 mg/ml; 89 ±27% 76 ±23%

PVl-46 3 2.29 ±3.41 mg/ml; 1.18 ±0.96 mg/ml 7.57 ±7.33 mg/ml; 86 ±14% 91 ±9%

PV1-50 1 1.01 mg/ml; 94% 0.44 mg/ml 11.22 mg/ml; 90%

PV1-57 1 0.67 mg/ml; 96% 0.22 mg/ml 11.88 mg/ml; 85%

PV1-76 1 1.32 mg/ml; 92% 0.04 mg/ml 11.88 mg/ml; 92%

PV22-32 1 0.04 mg/ml; 0% 4.88 mg/ml 6.31 mg/ml; 6%

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

Example 7 T cell response

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 x 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.

Results showed that the hybrids EA-PVl-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.

Table 4

Vespid antigen 5 or hybrid stimulated proliferation of murine spleen cells Spleen cells Stimulating Ag specific for EA-Ves v5 EA-Pol a 5 EA-hybrid

Stimulation Index at 100 μg/ml Ag

KR-Ves v 5 8.2 1.5 -

KR-Pol a 5 2.2 6.3 -

EA-PV1-155 6.1 2.2 5.0

EA-PVl-46 6.0 8.0 13.5

EA-PV1-18 2.3 5.0 6.1

EA-PV156-204 4.1 4.2 6.8

EA-PV195-204 1.7 8.6 4.1 μg/ml Ag for stimulation index of 4

KR-Ves v 5 2.6 >100 -

KR-Pol a 5 >100 16 -

EA-PV1-155 11 >100 0.54

EA-PVl-46 20 2.2 0.26

EA-PV1-18 >100 47 19

EA-PV156-204 60 70 2.3

EA-PV195-204 >100 8 82

Background proliferation of spleen cells showed ³H-thymidine uptake of 400-900 cpm.

Example 8 Allergenicity of recombinant vespid Ag 5s and hybrids in patients

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.

Table 5.

Summary of histamine release data of hybrids

Reciprocal Activity Relative to Ves v 5

Group A Group B

No. of No. of

Allergen patients Mean Range patients Mean Range

Ves v 5 7 1 1 3 1 1

Pol a 5 7 1154 330-5500 3 0.7 0.2 - 2

PV1-155 3 1 1-2 2 1 1

PVl- 46 5 126 13-3300 2 0.7 0.1 - 5

PV1- 18 3 583 12-5000 2 24 3.0 - 200 PV22-32 3 3207 2000-5000 2 6 6-20

PV115-125 3 3207 2000-5000 2 5 ^• 2-15

PV142-150 3 3000 2700-5000 2 5 2-15

PV156-204 6 1139 1000-3000 3 3 0.4 - 70

PV195-204 3 3207 50-5000 2 32 20.0 - 50

The complete data from one patient of each group are given in Figure 11. Of the three N-terminal hybrids tested, EA-PV1-155 showed no decrease in allergnenicity. EA-PVl-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.

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 Figure 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- teraiinal hybrid EA-PV156-204 as compared to the N-terminal hybrid EA-PVl-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, PVl-18 or PV195-204, as compared to their respective longer ones also reflects the influence of epitope density.

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). 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 PVl-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 PVl-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

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 incoφoration of heavy-atom derivatives and used to solve the three-dimensional stracture of Ves v 5. The atomic coordinates and stracture 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.

Table 6. Ves v 5 crystal coordinates

REMARK FI ENAME= "bref inement . pdb" REMARK r= 0 .215955 free r= 0.298202

REMARK DATE: 28-Oct-98 15:45: :46 created b y user: anette

ATOM 1 CB GLU 1 17. ,077 51.793 23. ,662 1.00 41. 80 APEP

ATOM 2 CG GLU 1 16. ,595 52.047 25. ,081 1.00 43. ,97 APEP

ATOM 3 CD GLU 1 15. .167 51.580 25. .310 1.00 44. ,74 APEP

ATOM 4 OE1 GLU 1 14. ,367 51.640 24, ,352 1.00 46. .38 APEP

ATOM 5 OE2 GLU 1 14. .845 51.156 26, .444 1.00 43, .48 APEP

ATOM 6 C GLU 1 19. ,169 50.429 23. ,664 1.00 39. ,72 APEP

ATOM 7 0 GLU 1 19. .733 49.575 24, .358 1.00 40. ,19 APEP

ATOM 8 N GLU 1 17. .005 49.431 24, .404 1.00 41, ,50 APEP

ATOM 9 CA GLU 1 17. .655 50.391 23, .458 1.00 40, .85 APEP

ATOM 10 N ALA 2 19. .820 51.423 23. .064 1.00 37, ,33 APEP

ATOM 11 CA ALA 2 21. .267 51.571 23, .179 1.00 34. .17 APEP

ATOM 12 CB ALA 2 21. .668 51.735 24 .657 1.00 34, .25 APEP

ATOM 13 C ALA 2 21 .935 50.341 22 .585 1.00 32, .32 APEP

ATOM 14 0 ALA 2 21. .299 49.580 21 .847 1.00 33, .01 APEP

ATOM 15 N GLU 3 23 .215 50.148 22 .899 1.00 29, .81 APEP

ATOM 16 CA GLU 3 23, .956 48.991 22 .402 1.00 26, .33 APEP

ATOM 17 CB GLU 3 24 .948 49.413 21 .325 1.00 30 .89 APEP

ATOM 18 CG GLU 3 25 .246 48.320 20 .303 1.00 35 .96 APEP

ATOM 19 CD GLU 3 24 .029 47.468 19 .973 1.00 38 .25 APEP

ATOM 20 OE1 GLU 3 23 .428 47.678 18 .891 1.00 39 .27 APEP

ATOM 21 OE2 GLU 3 23 .681 46.586 20 .793 1.00 37 .45 APEP ATOM 22 C • GLU 3 24.,693 48.,269 23.,530 1.00 21..89 APEP

ATOM 23 O GLU 3 25. ,780 48. ,679 23, ,959 1. ,00 20, ,16 APEP

ATOM 24 N ALA 4 24, ,093 47, ,180 23, .995 1. ,00 17, .32 APEP

ATOM 25 CA ALA 4 24. ,652 46. ,382 25, .080 1. .00 15, .71 APEP

ATOM 26 CB ALA 4 23. ,796 45. ,141 25. .302 1. ,00 12. .64 APEP

ATOM 27 C ALA 4 26. ,103 45. ,970 24. ,862 1. ,00 14, .17 APEP

ATOM 28 O ALA 4 26. ,816 45, ,710 25, .827 1. ,00 11, ,99 APEP

ATOM 29 N GLU 5 26. ,542 45. ,908 23, ,603 1. ,00 12. .66 APEP

ATOM 30 CA GLU 5 27, ,917 45. ,503 23, .319 1. ,00 13. .51 APEP

ATOM 31 CB GLU 5 28. ,222 45. ,583 21, ,817 1. ,00 15. ,08 APEP

ATOM 32 CG GLU 5 29. ,647 45. ,127 21, .479 1. ,00 20. ,49 APEP

ATOM 33 CD GLU 5 30. ,068 45. ,447 20. ,049 1. ,00 22. ,60 APEP

ATOM 34 OE1 GLU 5 29. ,224 45. ,948 19. ,278 1. ,00 24, ,69 APEP

ATOM 35 OΞ2 GLU 5 31. ,245 45, ,199 19. ,699 1. ,00 23, .87 APEP

ATOM 36 C GLU 5 28. ,949 46. ,339 24, ,065 1. ,00 12. ,46 APEP

ATOM 37 O GLU 5 30. ,025 45. ,847 24. .394 1. ,00 12, ,28 APEP

ATOM 38 N PHE 6 28. ,616 47. ,596 24. ,343 1. ,00 11, ,87 APEP

ATOM 39 CA PHE 6 29. ,546 48. ,491 25. ,022 1. 00 11. ,93 APEP

ATOM 40 CB PHE 6 29. ,459 49. 879 24. ,377 1. 00 12. ,32 APEP

ATOM 41 CG PHE 6 29. ,706 49. ,857 22. .887 1. 00 14. ,45 APEP

ATOM 42 CD1 PHE 6 28. .646 49. ,803 21. ,997 1. 00 14, ,86 APEP

ATOM 43 CD2 PHE 6 31. ,001 49. 811 22. ,381 1. 00 14. 25 APEP

ATOM 44 CE1 PHE 6 28. ,870 49. ,698 20. .623 1. 00 15. ,78 APEP

ATOM 45 CE2 PHE 6 31. ,236 49. ,705 21. .008 1. ,00 13. ,92 APEP

ATOM 46 CZ PHE 6 30. ,166 49. ,648 20, ,131 1. ,00 13, ,36 APEP

ATOM 47 C PHE 6 29. ,378 48. ,556 26. ,537 1. ,00 10. ,13 APEP

ATOM 48 O PHE 6 29. ,892 49. ,463 27, .201 1. ,00 9. ,26 APEP

ATOM 49 N ASN 7 28, ,658 47, ,568 27. .066 1. ,00 10, .89 APEP

ATOM 50 CA ASN 7 28. ,411 47. .422 28. .498 1. ,00 7, ,63 APEP

ATOM 51 CB ASN 7 27. ,040 46. ,786 28, .750 1. ,00 6, .94 APEP

ATOM 52 CG ASN 7 25, .897 47, .774 28, ,658 1, .00 5. .91 APEP

ATOM 53 OD1 ASN 7 26. ,049 48. ,953 28, ,962 1. ,00 6. ,68 APEP

ATOM 54 ND2 ASN 7 24, ,735 47, ,286 28, ,240 1. ,00 2, ,00 APEP

ATOM 55 C ASN 7 29, .477 46, ,428 28, ,929 1. ,00 8, ,03 APEP

ATOM 56 O ASN 7 29. .712 45. .448 28, .223 1. ,00 7, ,49 APEP

ATOM 57 N ASN 8 30, .126 46, .663 30. .066 1. ,00 7, ,97 APEP

ATOM 58 CA ASN 8 31, ,155 45, ,735 30, .536 1, ,00 9, .65 APEP

ATOM 59 CB ASN 8 32, .193 46, .469 31 .384 1, .00 11, .85 APEP

ATOM 60 CG ASN 8 33, .241 45. .531 31, .961 1, .00 13, .69 APEP

ATOM 61 OD1 ASN 8 33, .493 44, .459 31 .415 1, .00 12, .11 APEP

ATOM 62 ND2 ASN 8 33 .858 45', .935 33 .071 1, .00 12 .79 APEP

ATOM 63 C ASN 8 30 .553 44, .586 31 .350 1. .00 10, .91 APEP

ATOM 64 O ASN 8 30 .397 44 .690 32 .564 1. .00 11, .39 APEP

ATOM 65 N TYR 9 30 .225 43 .490 30 .674 1, .00 10, .20 APEP

ATOM 66 CA TYR 9 29 .631 42 .331 31 .328 1, .00 9, .11 APEP

ATOM 67 CB TYR 9 28 .956 41 .431 30 .287 1, .00 8 .55 APEP

ATOM 68 CG TYR 9 27 .727 42 .054 29 .689 1 .00 6 .89 APEP

ATOM 69 GDI TYR 9 27 .798 42 .805 28 .517 1 .00 8 .12 APEP

ATOM 70 CE1 TYR 9 26 .668 43 .423 27 .991 1 .00 9 .63 APEP

ATOM 71 CD2 TYR 9 26 .498 41 .932 30 .318 1 .00 7 .93 APEP

ATOM 72 CE2 TYR 9 25 .362 42 .543 29 .806 1 .00 9 .55 APEP

ATOM 73 CZ TYR 9 25 .452 43 .286 28 .646 1 .00 10 .64 APEP

ATOM 74 OH TYR 9 24 .325 43 .893 28 .149 1 .00 11 .41 APEP

ATOM 75 C TYR 9 30 .628 41 .509 32 .131 1 .00 10 .32 APEP

ATOM 76 O TYR 9 30 .237 40 .584 32 .840 1 .00 8 .46 APEP

ATOM 77 N CYS 10 31 .912 41 .834 32 .017 1 .00 11 .72 APEP

ATOM 78 CA CYS 10 32 .934 41 .098 32 .750 1 .00 13 .13 APEP tι σ\ D n ' H '>ιι VD r<ι cθ '* H i ι co _n σv i ι o ι r>] σι co o o n o o n cq σO^l Oo Oo NM fWi l<ΛΛ ωlD ^'Ψ <Ilι αcoi σO riH Mj rCni rnn ιnι Oo Hri Λ Mβ in c i ro i i r ro ro ro ro M N D IN O O O O O O O O O O O O O O o o o o o o o o o o o o o o HHH HH HH HH H H H H o H N Ch ro ro cΛ ∞ c^ N w o ^ m ι H i ro o ι H M m H ^ ^ ω H in w c^ ω ro ra ι c in ιn rø N ^ -^

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^ ιn r o ^ vo ιn ω vD vo o ι ιn o uι -o r!i m vo ω ι ro σι θ H H o ω ∞ ∞

* ιl1 ri n O ιl (<ι o l ^ * ^ o c^ n α ^ li) n ri ι)) * ll ri <ll n lIι a n H ri l ^ ul ^lι n u ri rt O\ n * f^ ri ι. ri ^ Io * ^o ri ι^ ι- ι<l l o n ^ ^B O (Λ ri M * ol M o ιn ιJ) ω ^|ι o l ι n M m o H tt1 ri ^I) ^^ιl H w * (n ^ n lJ1 ^ ιn Φ ^o ιn o N ^ o u) (^|l o a ^ ^ι) m ιϊι ιn ^ lJJ * α) (^ l!3 N O ln o^ (D o^ m fιl ^o m iϊι a m (1 ^ ffi ^D lJ: ol N Ul U) oo * ω α! o m α! ^ * M W r^ o\ lJ^ * ω ^ ^ ^ a ι) 0 ( a ιfl ln

H θ H o c C ^ ιn ^, ^ t) H H H o o σi H csι σ\ OT ro ι ι ∞ CΛ o o ω ι ιjι ^,* ^ M i) _'* m ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ro ^ ^ ro M M n r rn r ^ ^ co rn co ro iyi co ro m rn ro r rt m

N in m n ιo * ιn oι w oo n o <ι: ιn M (i N ^ ^ ri i3 Ch o\ vo ri θ iΛ co ri « N [>i h o n ^ tΛ (Λ ijι m o o ιιι « π a ιιι ιιι ri n * o ι>) rO l£> C^ ^ H O H O I -O ^ O rθ l CO O] ^ M σ O OO ^ σ ι ^ c ω O Ln -O N O ^ N in -^ co ιιι n θι ιjι co o n ri ιιi ι<) \o ψ iΛ n o ri 5\ ai H ι<ι ιn w ri iΛ !o o o o iD rιι ιn ψ (Λ θ » a n o ψ π ιιi ri iιi ιιι ιi) iD ^ m n * ιj w r ^ ^ H H H W fθ n vo o o cτι co ι iO i ω a3 CΛ co co co co cft cn o ι D i ιn

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(B O C ffl O Q H N rtl m O O Q _ « o Q ω N ffl O O P cq O Q W N ei u p u ω ≥i u u u υ υ t-i u p isi u u u u u u p B U U U U CJ g U O S CJ U P U CO 1-5 o u u u υ u o ia u u ϋij u a j o a u u o

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K) [ H !-■ M (-J KJ μ μ to ω μ H H H H H H H H H I→ ^ t-^| _-^, t-^, t t-^| t^< tr^< O O θ n θ O

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C ω ω <Λ> ω ω . ω ω ω ω ω . ω ω ω ω > > -o ω ω -υ ω > ω M

H M ι O O lH I-J W M ιJ ω W H M ω W W W |J> M ι( ω M ι^ O C0 lΛ 03 O ^ σi -J cυ m -j -^j ^ -o ω co o m -π H co c^ ω i^ ^ ^ υi σi -υ M ^ w ω co H to ω o -υ

ON •-j vo -J O i Lu σΛ Ui o ui i-j i-' o ω cD iπ co iΛJ c ∞ ω o o H ^ o o OJ ^ ^ 0 * 0 01 0l I J ~J U lO IΛ U μ * -J sJ * M ljl ιl> U W O O H W ω M * IΛ 01 1!) \O O U ^ O IΛ M J μ ^ O O M |ji ω ι|ι (ll l ιl> o «) U O ω ω LO Lj ω c ω ω ω ω ω ω ω ω ω J ω ω ω ω ω ω ω ω ^ ω i ω i^ ω ω ω H« M ω iN- ω ι υι cn σ\ Lπ uι o H ω ιi uι eΛ CD Co ^ -j oo ∞ o φ vo oo ^ σι Λ ιι^ uι o ^ ^ ^ κj φ i iΛi ii s] μ uι u> >ι ^ >π iJi uι u (n ^ >^ μ μ o oi ιi o M α u αι o ω o Φ ui ιi> μ u ^ o iJ ιπ ω ω t ιι> ιo v] <!i ιn ι ^

(_5 CO O O I ι CO CO H ^ ι^ CD ι^ W ι O O O in O O ] H H ^ ln H VO O lD ^ Ui α3 CO VO (^ o μ ω ^ Ni uι ^ ijι u αι κj n o ιs .j φ uι μ ιπ ιo Φ o iJ o (j] in <n αι o o ιi^] θ vi (iι o ιπ ^ μ ιn (n iΛ) (n o ι<ι θ j κ) μ ^ ^ ^ ιg ιπ κι ui ιι ιιι

^ uι * Ui m

H

M ιi) si t u o

H H H H H H H H H H t-' H l-ⁱ H H l-' l-' H H H M I-' l-' H I-' H H H H H H K H H H H H H H H H H H H H H H H I-^, l-' H I-^, l-> ooooooooooooooooooσoooooooooooooooooooooooooooooooooooooo ooooooooooooooooooσoooooooooooooooooooooooooooooooooooooo

H H ri ι'

CΛ -J ιJ -υ

ATOM 193 OH TYR 25 31.661 30.283 24.566 1.00 19,.74 APEP

ATOM 194 C TYR 25 32. 339 38. 060 23. 336 1. 00 18. ,24 APEP

ATOM 195 O TYR 25 31. 155 38. 404 23. 332 1. 00 17. ,58 APEP

ATOM 196 N GLY 26 33. ,340 38. 929 23. ,250 1. 00 19, .90 APEP

ATOM 197 CA GLY 26 33. ,086 40. 358 23. 182 1. 00 22. ,78 APEP

ATOM 198 C GLY 26 32. ,536 40. 927 21. ,886 1.oo 25, ,12 APEP

ATOM 199 O GLY 26 32. 260 42. 125 21. 815 1. 00 26. ,30 APEP

ATOM 200 N SER 27 32. ,362 40. 092 20. ,867 1. 00 26. ,19 APEP

ATOM 201 CA SER 27 31. ,855 40. ,570 19. ,583 1. 00 26, .72 APEP

ATOM 202 CB SER 27 32. ,960 40. 435 18. ,522 1. 00 25. ,95 APEP

ATOM 203 OG SER 27 32. ,457 40. ,041 17. ,259 1. 00 24, ,78 APEP

ATOM 204 C SER 27 30. ,586 39. ,839 19, ,139 1. ,00 26, ,86 APEP

ATOM 205 O SER 27 30. 159 38. 878 19. 774 1. 00 25. ,87 APEP

ATOM 206 N LEU 28 29. ,979 40. 312 18. ,053 1. 00 29, ,54 APEP

ATOM 207 CA LEU 28 28. 766 39. 695 17. 518 1. 00 30. ,96 APEP

ATOM 208 CB LEU 28 27. 793 40. 769 17. ,021 1. ,00 33, ,13 APEP

ATOM 209 CG LEU 28 28. ,127 42. 217 17. ,391 1. .00 34. .56 APEP

ATOM 210 CD1 LEU 28 29. 022 42. 812 16. ,319 1. 00 34. ,22 APEP

ATOM 211 CD2 LEU 28 26. 843 43. 030 17. ,551 1. ,00 34. ,12 APEP

ATOM 212 C LEU 28 29. 142 38. 769 16. 365 1. 00 30. 72 APEP

ATOM 213 O LEU 28 28. ,277 38. 224 15. ,673 1. 00 31. ,18 APEP

ATOM 214 N LYS 29 30. ,448 38. ,602 16. ,176 1. 00 30. ,29 APEP

ATOM 215 CA LYS 29 31. ,008 37. 759 15. ,124 1. 00 29. 17 APEP

ATOM 216 CB LYS 29 32. ,490 38. ,102 14. ,937 1. 00 31. ,20 APEP

ATOM 217 CG LYS 29 33. 016 37. 866 13. ,534 1. 00 32. 99 APEP

ATOM 218 CD LYS 29 34. ,528 37. ,785 13. ,521 1. 00 34. ,25 APEP

ATOM 219 CE LYS 29 35, ,150 39. ,121 13. .885 1. ,00 35. ,23 APEP

ATOM 220 NZ LYS 29 35, ,686 39. ,098 15. ,273 1. ,00 37. ,84 APEP

ATOM 221 C LYS 29 30. ,867 36. ,269 15. ,444 1. 00 27. ,53 APEP

ATOM 222 O LYS 29 31. ,446 35, ,772 16, ,413 1. ,00 25, .87 APEP

ATOM 223 N PRO 30 30, .104 35, .530 14, ,621 1, ,00 27, ,59 APEP

ATOM 224 CD PRO 30 29, ,362 36, ,011 13. ,442 1, ,00 26. ,03 APEP

ATOM 225 CA PRO 30 29, .905 34. .091 14, ,840 1, ,00 25, ,87 APEP

ATOM 226 CB PRO 30 28. .982 33, ,675 13, .694 1, ,00 25, ,48 APEP

ATOM 227 CG PRO 30 28, .330 34, .949 13, .245 1, ,00 24, .87 APEP

ATOM 228 C PRO 30 31 .182 33, .253 14 .871 1, .00 25, .41 APEP

ATOM 229 O PRO 30 32, .061 33, .404 14, .018 1, ,00 26, .47 APEP

ATOM 230 N ASN 31 31 .273 32 .376- 15 .866 1, .00 22 .04 APEP

ATOM 231 CA ASN 31 32, .407 31 .469 16 .030 1, .00 21 .43 APEP

ATOM 232 CB ASN 31 33 .061 31 .623 17 .413 1 .00 21 .58 APEP

ATOM 233 CG ASN 31 33 .840 32 .911 17 .564 1 .00 23 .13 APEP

ATOM 234 OD1 ASN 31 34 .581 33 .319 16 .672 1 .00 23 .71 APEP

ATOM 235 ND2 ASN 31 33 .680 33 .558 18 .713 1 .00 25 .47 APEP

ATOM 236 C ASN 31 31 .817 30 .071 15 .944 1 .00 19 .60 APEP

ATOM 237 O ASN 31 31 .743 29 .365 16 .948 1 .00 18 .51 APEP

ATOM 238 N CYS 32 31 .384 29 .667 14 .756 1 .00 18 .76 APEP

ATOM 239 CA CYS 32 30 .779 28 .348 14 .605 1 .00 18 .03 APEP

ATOM 240 C CYS 32 31 .690 27 .310 13 .975 1 .00 17 .09 APEP

ATOM 241 O CYS 32 31 .234 26 .464 13 .207 1 .00 13 .04 APEP

ATOM 242 CB CYS 32 29 .493 28 .456 13 .792 1 .00 17 .35 APEP

ATOM 243 SG CYS 32 28 .253 29 .528 14 .570 1 .00 16 .28 APEP

ATOM 244 N GLY 33 32 .974 27 .379 14 .311 1 .00 19 .59 APEP

ATOM 245 CA GLY 33 33 .942 26 .433 13 .786 1 .00 21 .31 APEP

ATOM 246 C GLY 33 33 .914 26 .269 12 .278 1 .00 22 .56 APEP

ATOM 247 O GLY 33 33 .985 27 .250 11 .532 1 .00 22 .89 APEP

ATOM 248 N ASN 34 33 .812 25 .021 11 .830 1 .00 22 .35 APEP

ATOM 249 CA ASN 34 33 .787 24 .724 10 .409 1 .00 23 .03 APEP iβ H iii o H ^ oi n iΛ N iΛ in iTi iΛ n iB o n o ^ o oi ui m ^ ui ri n ai H ft ri U O iii in ri ψ co ^ o iij o io o n ^ n o iΛ ri H i VtOi c 01i r Hi H 00 O r1 lJ^ ιtι ι * l ( lD lD ^ o ri n ul ri n ^ Ol ^ n <|ι ol ^ n ^ oo ^ι n ^^ * ιn ^ ^> ffl « ( H n ^D o o al fl lIl lo ^l) ( ⁽^ ^D O VO H O

O •* H o o H CΛ 0"i 00 O O H j r ^ Ln oi cn oo r- ># -θ i ι co co σι σ\ © ι o r^ o D ti -n o -n c^ oo t^ o σi o o H Ol Ol ro ro H H J ( H H ro ro ro

H H H H H H H H H H H H H H H H H H H H o ri lD H co N tD * ri n o n l « ri oi j α w ri * lJ (n ^ n ^ ri n o * Ol lJ m ri o ^ n ^ o ( ^ uι ^ ^ll ^ n H (l O ι<) ι^ p^ -l (Λ ^ ri Ol ^(l * ^^ ^ (Il t^ r^ lJ^ lJ^ l Ol O ^() r^ ^^ I n ll^ N o m o H c^ ιl <J o^ ιJl ( n ω ω n n ιB U) o oJ (J ^_ ιJl ^ Ol ω ιfl O ln o H ι7) « n ^ H <J m N H ^ n o u1 0 ln -) w > o ιn n ri m o o n o H ^ ω uι o ffi iιi « a * ιjι α) θ Λ <ji o o iJi ιi) θi ^ i!i ι. n o x io ώ <j ri cιι ri tι\ n H (o ιi) ι^,ι ^ ιi! CD r^ιι

^ ro ro ro re c^ H o θ σ» oo oo c c o m αD ro oo c i vo ι vo ι vo oo vo vo ^ ro o] H oι ro ro m

Ψ Ψ ^ ^f |ι ^ll ιι ιΛ W ιl^ ll ι^ u) ιfl ιn o a ω ω ω ^ ^ αJ lD m ω α) CD oo m <l m o o\ m o o o o o o o o o o o o ^l H ^^

W jj^< y^j2 W W W >i >i >i i ^ >i >i > e e e fi

H H OI H O] rt CV rt H V Ci moo _ pq O Q H N sϋ ffl O CD fJl CP O O (( B O O rtl CQ O rtJ ffl CD Q H Q W N U! <!

U U U P i-5 U U U U U !-3 U P suuuuupsguuuuupsuuuuupau upupiguuuuuuuupup iguu ul a lJ) (Λ O ri n n 'f ι -' ^ ((l 0^ o ri [<l ι<l ^ ιo «) ^ ι^ι ol O I-ι ^ι m ^ [n ^D ι ι ι^ N « M N C<i n M rJ r) W n N M r) n W N ( n ri N

Pε εPεPεPεP εPεP PεεPεPεPεP PεPsεPεPεP εPεPεPεPεPεPεPεP εPεP εPεP εPsPεP

IΛ r- a i-i ii ft i-i Pi ft ft ft ft iii ft i Pi i Pi ft Si ft ft i-i i Pi αi αi i-i ft ft ft Pi Pi ft ft ft αi ft i-i Pi ft ft ft vo p₄ Oι Oι Dι Dj ft Dj P₄ &ι ft p_J ft C-ι flι ι ι (-y α₄ (-j ιj Dj < α₄ C-_! (_q --, ς ft C £ f f^ ι< ι S rt; ι3 f3 rt; <; rt; C fs; ri: tf; C rt; ; ι rt; fcc £ f rt; ι3

Ln ^ⁱ C0 L0 l l 00 O H H 01 r0 O -n O I H Cft 0] O V0 O l V0 '* ri.. ri tΛ O ( N ^ MD O O\ * 0 ri H * ri OO H -φ ^ rO LO VO O H H O VO H O in m r0 00 ι-1 O CΛ σi -0 ^|ⁱ r0 V0 01 00 r0 V0 H CΛ 0] 00 r0 CΛ 00 l O tι Ul l l '* l '* 00 r)i H VO 0O H rO '* O] n o σ» cn cΛ H '*< CΛ oα ιji |i » H H vo

H U « fl « rl o 01 ro 01 ro H H rO Ol '^ 'φ VO CO σi H O rO '^ Ol O O Ol Ol H O σι σ\ σv co cn σι i o o o o σι σι ι CΛ H H O O H H H J Ol α. H H H H H H H H H H H H H H H H H H OI OI H H H H H H H H H -t ^ r-l H H H H H H H H H oooooooooooooooooooooooooooooooooσoo oooooo ooooooooooooo ooooooooooooooooooooooooooooooooooooooooooooooooooooooooo

HH HH H HH H HH H H HH H H H HH H H H HH H H H H H H H H H H H H H HH HH HH H H H H H H H

(Il O ln ^o o J ιn ri n u o (ll ^f ιf ιo H ri lΛ N (Λ n o n (n lo ^ o w n o ^|ι o) ^ ^^ (Λ o ^o m ιι) ιι o ^o ol l! o x r^ ^ cι -l N '* ιΛ ^o ri cιι a αι ^ (ri M ri ri ri ιn ii o » u) ι oo uι N iΛ m o * * u) ^ a co ui H o o tf flθ H U) co <|i ιιι ri m n (- -i o ri io ιn ^ m ro vo H vo ^ ι oι co oι oi M θi oι H CΛ co r H ^ Ln m σ^ ι co r oι r- vo oι m o ro o D ^ m

Oi rO rO ^ UO VD ^ Ol rO H H O 00 O) H Ol 01 Ol Ol H OI ΓO H H O O O O H l ro l ro Ol H O H m ^ ui n o kD iti i o o o in m 'iiⁱ H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

VO H H Ol CO CO n ln ιJl

01 o oo o in oi ^Il (Λ o H ι VD in ro co to o o (o ^) -l a m θ) II! «) ll! «l (ll i ro ro ro ro ro ro ro r r " * ι. ^ o H n ol ιf ri w ι. o « ω N Ol H (^ o ln ^ ιl O N n _l m ^ (ll N lo m ^ ^ ^ ^ ri a o n ln ^ oo ιJ^ m ^ ιo H ^ lD ^ (ll (i^ (Il ^ M u ιJ) W l ^ Bl U ιι o ri ιΛ t>l * o n ιΛ Ol a ιl n ιD ^o * ι<) tf ri m . ri co N O ri ^o ri c>l lI^ ^^ o o ^o . o ^^ ^^ n c^ ι)) Ol ln ul ^tι V Ol «l H ^ ^ c n ^ o n ^ H N N ι- lJ o ol m ι « ^ a o ri o! n o ι_ ω oo ol (^ o N ln n N ^ ω ι» ^^ι ^^ m '* H ^# o ^I! [ ^ c^l ^

^ » ι ιn w * ιn « ψ * n (>l ^ (1 M ^^ « ^^ o o (Λ o o o o ιI! ^) ^ l ιι^ * ^l o o ^^ ri l n N O lIl o o ^ι pl n ιn ^o ιn o^ (^) ιo I ψ

H H H H H H HHH H H HH H H H H H H HH H H H H H H H H H H H H H H H H H H H

H i oi oi oi w o i oi m ro r r ro ro ro ^ ^ ^ ^ ^ ^ ^ ^ ^ in i-i i in Ln ^ i ii Ln vo vo o vo vo ω

ι |-D t) & D |D & t3 D pi rt rt r-i Di rt « ω ιn w m ra ω cQ o. fe g U ,-q j ₁J ι-3 ι4 .J ιJ J EH H H EH -H H H J .4 J H ι >-P .J .J ι^

H Ol H Ol rl H Ol s rtυJuCQ UυuPuW Nsuofe f H O O i-q u u u u υ u p g u u o u u o υduuuHpHsuoB rtuJυm OuQupH Wp u riJ pq O ^Q H N I ffl O ^Q

P I-5 U U U U U I-5 U P I-5 U U U U

IΛ Φ Λ O H N π * ι1 ^ l- m θ H W n ^ m « IJ) 01 0 H N n 'φ U1 \0 m 01 0 ri (S n <J W 10 IIl CΛ O ri N m '* in i!) O (Λ O rl (>i vo © O o θ H H H H H H H H H H θi oι oι ι oι ι r^ oι oι oι ro ro r ι ι ro ro r ro r o ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ -n ιΛ -π -n ι-i ι ιn -n ιn m r- ro ro rO ro ro ro ro ro ro ro ro ro M io ro rO ro ro ro ro ro r ro ro ro ro ro ro rO ro rO ir) ro ro r ro ro ro r^

©

© o ε ε ε s ε ε ε ε ε ε ε ε ε ε ε ε ε ε ε s ε ε ε ε ε s ε ε ε ε ε ε ε ε ε ε ε ε s ε ε ε ε ε ε ε ε ε ε ε ε s ε ε ε ε s P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P P O P P P P P P P P P P P P P P P P P P

H H i ι EH E-' B H H EH H H -H H H H B E-ι H --H EH H H H ι>" H l ι

> ; C ι l C ^ ι: ι^ C C C d ι^ C '<! C ι^ ' l ι l ' l d ^

ATOM 36-4 OEl GLN 48 5.,301 44.,606 14.,359 1.00 10.,04 APEP

ATOM 365 NE2 GLN 48 3. ,758 43. ,065 13. .816 1. ,00 11, ,92 APEP

ATOM 366 C GLN 48 6, .420 40. ,123 16, ,563 1, ,00 12, ,69 APEP

ATOM 367 O GLN 48 5. .488 39. ,993 17. ,353 1. ,00 13, ,53 APEP

ATOM 368 N ASP 49 6. .716 39. ,219 15. ,633 1. ,00 12. ,63 APEP

ATOM 369 CA ASP 49 5. .964 37. ,977 15. .487 1. ,00 11. ,04 APEP

ATOM 370 CB ASP 49 6, .290 37. ,322 14. ,144 1. ,00 14, ,99 APEP

ATOM 371 CG ASP 49 5. ,578 37. ,990 12, .981 1. ,00 17. ,72 APEP

ATOM 372 OD1 ASP 49 4, ,518 38. ,620 13, ,200 1. ,00 18, ,74 APEP

ATOM 373 OD2 ASP 49 6, .082 37. .878 11. ,844 1. ,00 19, ,80 APEP

ATOM 374 C ASP 49 6. ,285 36. ,998 16. ,615 1. 00 9. ,65 APEP

ATOM 375 O ASP 49 5. ,433 36. ,211 17. .020 1. ,00 9, ,33 APEP

ATOM 376 N ILE 50 7. ,519 37. ,034 17. ,107 1. ,00 8, ,25 APEP

ATOM 377 CA ILE 50 7, ,916 36. ,152 18, ,203 1. ,00 8. ,01 APEP

ATOM 378 CB ILE 50 9, ,454 36, ,132 18, ,387 1. ,00 7. ,72 APEP

ATOM 379 CG2 ILE 50 9, .823 35. ,416 19. ,693 1. ,00 7. ,19 APEP

ATOM 380 CGI ILE 50 10, ,103 35. ,410 17. ,203 1. ,00 6. ,44 APEP

ATOM 381 CD1 ILE 50 11. ,582 35. ,687 17. ,041 1. oo 4. ,97 APEP

ATOM 382 C ILE 50 7, ,256 36. ,621 19. ,499 1. ,00 8. ,14 APEP

ATOM 383 O ILE 50 6, ,805 35. ,808 20. ,303 1. ,00 7. ,29 APEP

ATOM 384 N LEU 51 7, ,191 37. ,938 19. ,679 1. ,00 8, ,57 APEP

ATOM 385 CA LEU 51 6, ,571 38. ,529 20. ,854 1. 00 9. 76 APEP

ATOM 386 CB LEU 51 6. ,733 40, ,055 20. ,836 1. ,00 9. 57 APEP

ATOM 387 CG LEU 51 6, ,509 40, ,844 22. ,139 1. ,00 12. ,08 APEP

ATOM 388 CD1 LEU 51 7, ,509 40, ,401 23. ,216 1. ,00 9. ,69 APEP

ATOM 389 CD2 LEU 51 6, ,659 42, ,333 21, ,861 1. ,00 10. ,85 APEP

ATOM 390 C LEU 51 5. ,091 38, ,172 20, ,863 1. ,00 10, .95 APEP

ATOM 391 O LEU 51 4, .571 37. ,664 21. ,861 1. ,00 12. ,16 APEP

ATOM 392 N LYS 52 4, ,423 38, ,427 19. ,739 1. ,00 10, ,41 APEP

ATOM 393 CA LYS 52 2, .994 38, .156 19. ,601 1, ,00 10, ,29 APEP

ATOM 394 CB LYS 52 2, .520 38, .535 18. .196 1, .00 10, .94 APEP

ATOM 395 CG LYS 52 1, ,066 38, ,956 18, ,132 1. ,00 14. ,35 APEP

ATOM 396 CD LYS 52 0, .258 38, .029 17, ,236 1. ,00 16, ,34 APEP

ATOM 397 CE LYS 52 -0, .870 38, .780 16, .543 1, .00 17, ,57 APEP

ATOM 398 NZ LYS 52 -2, .107 38, .817 17, .374 1, .00 17, ,77 APEP

ATOM 399 C LYS 52 2 .627 36, .709 19, .893 1, .00 9, .70 APEP

ATOM 400 O LYS 52 1 .553 36 .432 20 .419 1, .00 9, .63 APEP

ATOM 401 N GLU 53 3 .508 35 .780 19 .540 1, .00 10, .85 APEP

ATOM 402 CA GLU 53 3 .249 34, .366 19, .799 1, .00 11. ,95 APEP

ATOM 403 CB GLU 53 4 .261 33 .491 19 .057 1, .00 13, .57 APEP

ATOM 404 CG GLU 53 3 .957 31 .996 19 .089 1 .00 15 .51 APEP

ATOM 405 CD GLU 53 2 .525 31 .651 18 .695 1 .00 20 .29 APEP

ATOM 406 OEl GLU 53 1 .876 32 .439 17 .971 1, .00 21, .72 APEP

ATOM 407 OE2 GLU 53 2 .044 30 .577 19 .111 1 .00 21 .86 APEP

ATOM 408 C GLU 53 3 .362 34 .120 21 .294 1 .00 12 .09 APEP

ATOM 409 O GLU 53 2 .568 33 .382 21 .878 1 .00 11 .99 APEP

ATOM 410 N HIS 54 4 .357 34 .750 21 .910 1 .00 12 .11 APEP

ATOM 411 CA HIS 54 4 .580 34 .610 23 .340 1 .00 11 .34 APEP

ATOM 412 CB HIS 54 5 .829 35 .399 23 .769 1 .00 9 .29 APEP

ATOM 413 CG HIS 54 7 .089 34 .584 23 .817 1 .00 7 .76 APEP

ATOM 414 CD2 HIS 54 7 .695 33 .933 24 .840 1 .00 9 .61 APEP

ATOM 415 NDI HIS 54 7 .895 34 .394 22 .716 1 .00 6 .60 APEP

ATOM 416 CE1 HIS 54 8 .941 33 .663 23 .056 1 .00 5 .37 APEP

ATOM 417 NE2 HIS 54 8 .844 33 .370 24 .340 1 .00 7 .30 APEP

ATOM 418 C HIS 54 3 .365 35 .143 24 .092 1 .00 10 .89 APEP

ATOM 419 O HIS 54 2 .844 34 .492 24 .983 1 .00 10 .22 APEP

ATOM 420 N ASN 55 2 .913 36 .331 23 .703 1 .00 12 .43 APEP ATOM 421 CA^* ASN 55 1.,784 36.982 24.356 .00 13.65 APEP

ATOM 422 CB ASN 55 1..791 38.473 23.991 ,00 12.77 APEP

ATOM 423 CG ASN 55 2..950 39.232 24.655 .00 12.31 APEP

ATOM 424 OD1 ASN 55 3, .396 38.871 25.747 00 7.53 APEP

ATOM 425 ND2 ASN 55 3.436 40.280 23.993 00 9.40 APEP

ATOM 426 C ASN 55 0.413 36.347 24.097 .00 13.82 APEP

ATOM 427 O ASN 55 -0.457 36.355 24.973 .00 13.31 APEP

ATOM 428 N ASP 56 0..221 35.795 22.907 .00 12.69 APEP

ATOM 429 CA ASP 56 -1, .036 35.134 22.572 .00 12.59 APEP

ATOM 430 CB ASP 56 -1..049 34.675 21.111 .00 12.80 APEP

ATOM 431 CG ASP 56 -1.359 35.787 20.143 1.00 13.37 APEP

ATOM 432 OD1 ASP 56 -1. .902 36.825 20.565 1.00 13.48 APEP

ATOM 433 OD2 ASP 56 -1. .059 35.615 18.945 1, 00 15.39 APEP

ATOM 434 C ASP 56 -1, .153 33.899 23.450 1, 00 10.48 APEP

ATOM 435 O ASP 56 -2, .224 33.577 23.953 1, 00 10.14 APEP

ATOM 436 N PHE 57 -0. .046 33.191 23.604 1, 00 9.15 APEP

ATOM 437 CA PHE 57 -0, .047 31.989 24.418 1, 00 11.09 APEP

ATOM 438 CB PHE 57 1.272 31.227 24.252 1.00 12.68 APEP

ATOM 439 CG PHE 57 1.261 29.863 24.884 1.00 11.85 APEP

ATOM 440 CD1 PHE 57 0.346 28.899 24.471 1.00 12.69 APEP

ATOM 441 CD2 PHE 57 2.150 29.549 25.903 1.00 12.28 APEP

ATOM 442 CE1 PHE 57 0.316 27.642 25.067 1.00 11.80 APEP

ATOM 443 CE2 PHE 57 2.132 28.296 26.508 1.00 12.07 APEP

ATOM 444 CZ PHE 57 1.216 27.342 26.092 1.00 12.36 APEP

ATOM 445 C PHE 57 -0.267 32.346 25.890 1.00 10.66 APEP

ATOM 446 O PHE 57 -1, .012 31.664 26.597 1.00 11.28 APEP

ATOM 447 N ARG 58 0, .360 33.423 26.349 1.00 8.83 APEP

ATOM 448 CA ARG 58 0 ..204 33.830 27.738 1.00 10.25 APEP

ATOM 449 CB ARG 58 1, .107 35.024 28.057 1. 00 7.50 APEP

ATOM 450 CG ARG 58 2 .483 34.615 28.530 1.00 7.56 APEP

ATOM 451 CD ARG 58 3 .478 35.755 28.446 1, 00 7.29 APEP

ATOM 452 NE ARG 58 3 .391 36.649 29.601 1, 00 8.58 APEP

ATOM 453 CZ ARG 58 4 .025 36.450 30.750 1, 00 7.65 .APEP

ATOM 454 NH1 ARG 58 4. .797 35.388 30.908 1, 00 8.61 APEP

ATOM 455 NH2 ARG 58 3 .892 37.318 31.738 1.00 9.84 APEP

ATOM 456 C ARG 58 -1.246 34.170 28.032 1.00 9.76 APEP

ATOM 457 O ARG 58 -1.793 33.733 29.042 1.00 11.57 APEP

ATOM 458 N GLN 59 -1.874 34.932 27.141 1 00 10.78 APEP

ATOM 459 CA GLN ' 59 -3.270 35.321 27.314 1 00 9.25 APEP

ATOM 460 CB GLN 59 -3.621 36.464 26.368 1 00 11.30 APEP

ATOM 461 CG GLN 59 -3 .388 37.870 26.937 1 00 14.86 APEP

ATOM 462 CD GLN 59 -3 .254 37.924 28.457 1 00 15.40 APEP

ATOM 463 OEl GLN 59 -2 .306 38.508 28.976 1 00 20.39 APEP

ATOM 464 NE2 GLN 59 -4 .,203 37.328 29.171 1 00 16.19 APEP

ATOM 465 C GLN 59 -4.233 34.156 27.107 1 00 8.85 APEP

ATOM 466 O GLN 59 -5.275 34.084 27.753 1 00 8.65 APEP

ATOM 467 N LYS 60 -3.900 33.240 26.209 1 00 9.28 APEP

ATOM 468 CA LYS 60 -4.765 32.084 25.999 1 00 9.29 APEP

ATOM 469 CB LYS 60 -4.190 31.170 24.919 1 00 11.77 APEP

ATOM 470 CG LYS 60 -5.097 29.999 24.555 1.00 12.61 APEP

ATOM 471 CD LYS 60 .357 28.678 24.660 1.00 12.86 APEP

ATOM 472 CE LYS 60 .849 28.205 23.310 ,00 10.33 APEP

ATOM 473 NZ LYS 60 .535 26.970 22.830 .00 12.74 APEP

ATOM 474 C LYS 60 .840 31.329 27.320 00 9.51 APEP

ATOM 475 O LYS 60 .922 31.017 27.816 00 7.72 APEP

ATOM 476 N ILE 61 .671 31.049 27.887 00 9.04 APEP

ATOM 477 CA ILE 61 .570 30.348 29.161 00 9.87 APEP ATOM 478 CB ILE 61 -2.,075 30.,101 29.,536 1.00 10.78 APEP

ATOM 479 CG2 ILE 61 -1. ,970 29. ,598 30. ,973 1. 00 10. 13 APEP

ATOM 480 CGI ILE 61 -1. 428 29. 152 28. ,507 1. 00 9. 94 APEP

ATOM 481 CD1 ILE 61 -1. 212 27. 720 28. ,980 1. 00 10. 64 APEP

ATOM 482 C ILE 61 -4. .254 31. ,141 30, ,283 1. ,00 9. ,04 APEP

ATOM 483 O ILE 61 -4. ,980 30. ,571 31, ,092 1. ,00 9. ,20 APEP

ATOM 484 N ALA 62 -4. ,041 32. 454 30. ,314 1. 00 8. ,91 APEP

ATOM 485 CA ALA 62 -4. 628 33. 308 31. ,350 1. 00 9. 06 APEP

ATOM 486 CB ALA 62 -4. 090 34. ,729 31. ,209 1. 00 5. 84 APEP

ATOM 487 C ALA 62 -6. ,165 33. 327 31, ,363 1. 00 11. ,19 APEP

ATOM 488 O ALA 62 -6. 794 33. 581 32. ,397 1. 00 12. 79 APEP

ATOM 489 N ARG 63 -6. 769 33. ,053 30. ,214 1. 00 12. 40 APEP

ATOM 490 CA ARG 63 -8. 219 33. 050 30. ,096 1. 00 10. 93 APEP

ATOM 491 CB ARG 63 -8. 618 33. ,627 28. ,736 1. 00 10. ,77 APEP

ATOM 492 CG ARG 63 -8. ,043 35. ,012 28, ,505 1. 00 12. ,79 APEP

ATOM 493 CD ARG 63 -8. ,608 35. ,684 27. ,278 1. 00 15. , 66 APEP

ATOM 494 NE ARG 63 -7. ,868 36. ,904 26. ,968 1. 00 17. 96 APEP

ATOM 495 CZ ARG 63 -7. ,346 37. ,179 25. ,777 1. 00 20. ,00 APEP

ATOM 496 NH1 ARG 63 -7. ,483 36. ,321 24. ,772 1. 00 19. ,36 APEP

ATOM 497 NH2 ARG 63 -6. 679 38. 313 25. ,590 1. 00 22. 72 APEP

ATOM 498 C ARG 63 -8. ,827 31. ,661 30. ,285 1. 00 10. 92 APEP

ATOM 499 O ARG 63 10. .036 31. ,489 30, ,179 1. 00 12. 37 APEP

ATOM 500 N GLY 64 -7. ,986 30. ,677 30. .575 1. 00 12. 22 APEP

ATOM 501 CA GLY 64 -8. ,475 29. ,325 30, ,780 1. 00 11. 71 APEP

ATOM 502 C GLY 64 -8. ,985 28. ,685 29, .509 1. ,00 13. ,26 APEP

ATOM 503 O GLY 64 -9. 950 27. 911 29. ,540 1. 00 14. 03 APEP

ATOM 504 N LEU 65 -8, ,331 28. ,998 28, ,391 1. ,00 11. ,24 APEP

ATOM 505 CA LEU 65 -8. ,711 28, ,463 27, ,095 1. ,00 10. ,84 APEP

ATOM 506 CB LEU 65 -8. ,747 29. ,581 26. ,044 1. 00 10. ,48 APEP

ATOM 507 CG LEU 65 -9. ,602 30. ,803 26, ,396 1. 00 8. ,01 APEP

ATOM 508 CD1 LEU 65 -9, ,278 31. ,946 25, ,470 1. ,00 13. ,03 APEP

ATOM 509 CD2 LEU 65 11, .074 30. .450 26, .291 1. .00 10, ,77 APEP

ATOM 510 C LEU 65 -7, .764 27. .361 26, .644 1. ,00 12. ,39 APEP

ATOM 511 O LEU 65 -7. ,998 26, ,719 25, .625 1. .00 12. ,90 APEP

ATOM 512 N GLU 66 -6, .686 27, .147 27, .387 1, .00 11. ,27 APEP

ATOM 513 CA GLU 66 -5, .754 26, .094 27, .023 1, .00 12, .09 APEP

ATOM 514 CB GLU 66 -4 .365 26 .361 27 .610 1, .00 11 .39 APEP

ATOM 515 CG GLU 66 -3, .327 25, .308 27 .245 1, .00 12, .91 APEP

ATOM 516 CD GLU 66 -3 .362 24, .941 25 .774 1, .00 13 .85 APEP

ATOM 517 OEl GLU 66 -2 .689 25 .629 24 .988 1 .00 18 .33 APEP

ATOM 518 OE2 GLU 66 -4 .054 23 .971 25 .401 1 .00 12 .27 APEP

ATOM 519 C GLU 66 -6, .323 24 .799 27 .575 1, .00 12 .38 APEP

ATOM 520 O GLU 66 -6 .214 24 .512 28 .764 1 .00 12 .85 APEP

ATOM 521 N THR 67 - 6 .943 24 .022 26 .696 1 .00 13 .75 APEP

ATOM 522 CA THR 67 -7 .553 22 .769 27 .091 1 .00 13 .16 APEP

ATOM 523 CB THR 67 -8 .562 22 .295 26 .018 1 .00 13 .92 APEP

ATOM 524 OG1 THR 67 -7 .858 21 .894 24 .830 1 .00 14 .81 APEP

ATOM 525 CG2 THR 67 -9 .524 23 .413 25 .671 1 .00 11 .42 APEP

ATOM 526 C THR 67 -6 .548 21 .652 27 .368 1 .00 13 .63 APEP

ATOM 527 O THR 67 -6 .875 20 .682 28 .049 1 .00 14 .84 APEP

ATOM 528 N ARG 68 -5 .326 21 .793 26 .861 1 .00 13 .12 APEP

ATOM 529 CA ARG 68 -4 .301 20 .770 27 .042 1 .00 11 .47 APEP

ATOM 530 CB ARG 68 -3 .222 20 .914 25 .967 1 .00 13 .83 APEP

ATOM 531 CG ARG 68 -3 .715 20 .741 24 .538 1 .00 13 .10 APEP

ATOM 532 CD ARG 68 -2 .626 21 .128 23 .542 1 .00 13 .89 APEP

ATOM 533 NE ARG 68 -2 .317 22 .556 23 .590 1 .00 12 .94 APEP

ATOM 534 CZ ARG 68 -1 .244 23 .111 23 .033 1 .00 11 .20 APEP IΛ pi fr P^j. Q. ς^jj. fr fr C & P. Cjj. Α Α Α Α fr & P' l-. Qi. Α r- vo p^j. r-t & ft ft & e Pι ft ft < & ft P & ft ft & Pι ft & & vo ι ^ ^■* H ro '* m ^^ u) ω vo ^ ω o ri ^ o H n o ω ^ ^Λ lo H o ^ ^ ri π o * cD H -l ^,* <Λ lrl ^o ln <τι (o ^>l r^ ^ (O ιn ^ ι^,l ιn o lι^ in ω r- 01 vo 01 ^ o ( O ^Iι π ω o n N n ^o l O co ^o o ^^l rt '* ιIl ιι^ ι^,^ -l ^^ (^,l M O ιΛ ι# -l ^ ψ cIl (l r^ * ι^l αl <!^ ^ n <D *

H H O H cri rsi o oi O O O O 01 O H ro ro Ol ^■* •* ^■■# U H H H H H H H H H H H H H H H H H H α. ooooooooooooooooooooooooooooooooooooooooooooσoooooooooooo oσooooooooooooooooooooooooooooooooooooooooooσoooooooooooo

H H H H H H H H H H H H H H H HH H HH H H H H H H H H H H H HH HH H rl <-l - H HH H H H H H H H H rl ι) lJ^ n m ω ^l ri ^ * o (^ ω w (n vo * H o r^ n ω n ιn rt ^ ιo n ( a3 a) vo n n ^ ^ ^ o m ri ri N ri m ^ o ^^ ι l o o ιϊ) (^ ιI n H rη vo ^ ri * a3 m (η σl ^ ( n π ^o ω ri <) (o m ^ H m σ^ w o ^ ri ^J ^o m o ^ ^ N » m ^ ^ o M m o ri ^ ^ ω Il ^o >!tl {η ri ra n H ^ o o ri ι^l ιo ri ri n ιJ) ι» ul ιι^ o Φ O ^ <f n l1 ri ri ^ N ri (Λ H ri ^^ ^o ιι^ a o o o lO r^ lll <|l N O ιι1 ln ^l! ^ι n n |ι ι^l n ι<l

N n a) (Λ o o o ij> ri n n oi ri n n * n ιn N n * n tι ri H H i ri N ri θ M n n ri N ri H « n H n M ι ιjι ι « ιo ^ * * ιn * ^ ^ ιn ιi) ol ol Ol ol ol ro ro ol ro ro ro ro ro ro ro ro ro ro rθ ι?o ro ro ro ιτ> ro ro ro ro ro rό ro ro ro ro ro ro ro ro ro ro ro

H |Il o ^lJ (n ω o ι» ι- O ri ln ^|ι ^ι n -ι ^ ui n H n n iιi ι^,) '* * iιi iΛ θ i⁾i '* θ (D n ι» N ri ri ^ H lJl ln ιn lll n d ^ p^ ιJ^ ol oι

Ol Ol Ol Ol Ol Ol Ol Ol Ol Ol Ol Ol Ol Ol rO rO rO ro rO

01 0] H O Ln H -n C0 VD V0 01 O '* V0 01 l O I r 01 00 01 C0 V0 CM ι O O rO Ol H OI Ol H rO Ol OO OO O tj co i l o ro θ '4' Vo o r- ι r ^H co r '5iι o ι ι - ro oι o jι vo oι o o ro H o o ιn H i vo H VD oι oi ro vθ 'jι ro ro o ^ ri Ol ri ri r- H OO O LO ι Lθ ro o H θi ro -n r- ro ι H oι vo vo co co o [-~ rθ O V0 ^ 01 VD in CΛ O CO '* rθ 01 CO '5t^, l l '^ι l 01 01 01 VO VO O J< 0 0 ro ^ vθ Lo -* oι ι ι ι vo o " '-n" ri n o rg to ^ o o ri ψ iΛ m n o ii) o H ro ro n oi oi oi oi O O H O M ri n ^ ^ ιfl ul ^ « w ^o aJ 03 m ^ a lJ^ ω o ιll * m M r^ ri ^ι ιl ω ιι^ ^# o ιϊ) * ^D _) oo Il -) lIι £ι ιn

I I I I I I I I I i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i i

to o ai iD Λ iΛ in oi o o o o o o o o ri ri H ri ri H H N n N n n rt n n c r n ^ ψ ψ ψ ^ ^ ^ ^ n i in iri io in -i io io ^ iD io io iD ^l) ^o o u) ^o «ι «) ι. ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^

q o o o iH SH iH iH ig ig g ig ig isi g ig o o o o o o o ix ^ iH iH O o o o o o o g i-i izi a ig iz is s a O _ P _ P ^ P _, P ^ P _ P _, P ^ P _, P _, p _, Q ^ P _, P _ # 2 # 2 ^ j ij i-} ra to TO w w w w cQ P, tf tf pS tf θ O O O ^ < < < < h h Pι |^] _J p_l ft a θ θ Q θ iiι p_l |i_< Pι Pι |iι |iι θ θ O θ θ O Q [!) 0 &

CM H Ol ei ri! cq σ Q Q rtJ m o «: Q rti m riJ ffl CD Q H W Q rt! m θ P fϋ ffl O

U P a u u o g u u u p U P S U U U U U P S U U O i-S U U U U U p tg U U U U P S U P S U U U U U P S U U U U U O

ATOM 522 N ' ALA 77 5 .553 32.697 34.930 1.00 15 36 APEP

ATOM 593 CA ALA 77 4 . 617 33.458 35.745 1 .00 16 81 APEP

ATOM 594 CB ALA 77 264 33.531 35.050 1 .00 15 29 APEP

ATOM 595 C ALA 77 111 34.864 36.034 1 .00 18 21 APEP

ATOM 596 O ALA 77 946 35.414 35.315 1 .00 18 77 APEP

ATOM 597 N LYS 78 578 35.447 37.095 1 .00 19 69 APEP

ATOM 598 CA LYS 78 944 36.799 37.487 1 .00 21 45 APEP

ATOM 599 CB LYS 78 5 .453 36.779 38.934 1 .00 19 28 APEP

ATOM 600 CG LYS 78 5 .408 38.109 39.658 1 .00 21 07 APEP

ATOM 601 CD LYS 78 5 . 939 37.969 41.078 1 .00 22 93 APEP

ATOM 602 CE LYS 78 7 . 039 38.993 41.380 1 .00 22 07 APEP

ATOM 603 NZ LYS 78 8 .416 38.442 41.192 , 00 18. 83 APEP

ATOM 604 C LYS 78 3 .681 37.655 37.351 , 00 20. 82 APEP

ATOM 605 O LYS 78 -3.735 38.825 36.973 , 00 22 .49 APEP

ATOM 606 N ASN 79 -2.546 37.024 37.632 , 00 20. 62 APEP

ATOM 607 CA ASN 79 -1.225 37.650 37.596 1 . 00 20. 96 APEP

ATOM 608 CB ASN 79 -0.322 36.893 38.591 1.00 21.73 APEP

ATOM 609 CG ASN 79 0.895 37.696 39.041 1 . 00 26.41 APEP

ATOM 610 OD1 ASN 79 1.739 37.194 39.794 1. 00 27.31 APEP

ATOM 611 ND2 ASN 79 0.995 38.941 38.586 1, 0 000 30 92 APEP

ATOM 612 C ASN 79 -0.570 37.649 36.199 1, 0 000 20 23 APEP

ATOM 613 O ASN 79 0.658 37.679 36.109 1, 00 20 51 APEP

ATOM 614 N MET 80 1. 354 37.648 35.117 1, 00 17 31 APEP

ATOM 615 CA MET 80 0 .745 37.581 33.783 1 00 16 95 APEP

ATOM 616 CB MET 80 1.334 36.398 33.014 1 00 14 01 APEP

ATOM 617 CG MET 80 0 .475 35.941 31.848 1 00 10 95 APEP

ATOM 618 SD MET 80 001 35.032 32.360 1 0 000 10 66 APEP

ATOM 619 CE MET 80 309 33.392 32.631 1. 0 000 9 65 APEP

ATOM 620 C MET 80 743 38.809 32.863 1 00 17 13 APEP

ATOM 621 O MET 80 785 39.236 32.377 1 00 17 76 APEP

ATOM 622 N LYS 81 450 39.343 32.602 1 00 16 19 APEP

ATOM 623 CA LYS 81 621 40.509 31.734 1 00 15 79 APEP

ATOM 624 CB LYS 81 360 41.626 32.480 1 00 19 28 APEP

ATOM 625 CG LYS 81 485 42.479 33.376 1 00 23 64 APEP

ATOM 626 CD LYS 81 1.283 42.976 34.581 1 00 29 37 APEP

ATOM 627 CE LYS 81 0 . 652 42.551 35.914 1 00 30 79 APEP

ATOM 628 NZ LYS 81 1 . 639 41.850 36.794 1 00 31 43 APEP

ATOM 629 C LYS 81 1.428 40.135 30.489 1 00 15 20 APEP

ATOM 630 O LYS 81 2 . 144 39.133 30.478 1 00 13 27 APEP

ATOM 631 N ASN 82 1.317 40.947 29.445 1 00 14 40 APEP

ATOM 632 CA ASN 82 2.047 40.695 28.214 1 00 14 06 APEP

ATOM 633 CB ASN 82 1.442 41.492 27.059 1 00 15 .84 APEP

ATOM 634 CG ASN 82 0 . 081 40.970 26.636 1 00 19 .06 APEP

ATOM 635 OD1 ASN 82 0 . 837 41.746 26.366 1 00 20 .37 APEP

ATOM 636 ND2 ASN 82 0. 058 39.649 26.579 1 00 20 55 APEP

ATOM 637 C ASN 82 3 .496 41.107 28.400 1.00 11.87 APEP

ATOM 638 O ASN 82 3 . 800 41.968 29.226 1.00 11.67 APEP

ATOM 639 N LEU 83 4 . 384 40.483 27.633 00 10.14 APEP

ATOM 640 CA LEU 83 5 . 809 40.789 27.684 00 9.10 APEP

ATOM 641 CB LEU 83 6 . 648 39.577 27.272 00 9.30 APEP

ATOM 642 CG LEU 83 6.373 38.230 27.935 00 9.01 APEP

ATOM 643 CD1 LEU 83 7 . 077 37.119 27.178 00 10.20 APEP

ATOM 644 CD2 LEU 83 6.843 38.283 29.378 00 10.62 APEP

ATOM 645 C LEU 83 6.104 41.919 26.718 00 7 69 APEP

ATOM 646 O LEU 83 5.285 42.254 25.866 00 7 01 APEP

ATOM 647 N VAL 84 7.277 42.516 26.878 00 7 90 APEP

ATOM 648 CA VAL 84 7.736 43.585 26.004 00 9 09 APEP IΛ r- vo iA ii ft ft & α) P₎ o₎ p< p₎ ft pj. pj r-) P< ft θι & r-) ft ft ft ft r ri! ι< r3 ι< <! ι3 ι< r ι< r< ι< sC ,< ,< ;< f ι ι< .4 o o L0 VD C0 C0 '* 00 V0 V0 I r0 L0 01 Ln -0 l O i ro ro i ro VD VD ι θD ^ H ro ιTι ιn ro i co r H oo oD Cil o ^ oi H ι-l ^ ro ro cι oι vo ι cιl H r '> vo >* oo H H O og r- o o oi vo o vo H oo co r- H o ro ι oι ^ vo H C o ι cΛ iJi H H m r^ ro N C oo o σι o ^ oι vo o vo ^ cι m ι co ι co r

H C¹1 H ω ro t~ Cι1 αD l J Vo ω i-1 Vθ m LO r rθ 01 01 CΛ 03 ^ ιJ1 LO VO i VO -O Ci1 0 01 0 H H 01 0 01 01 01 r 01 01 CO CO t- l VO -n l r- co 01 D 01 o U H H H H H H H H H H α. o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o σ o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o o

H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H ιn oι r σ> vo r H r r cM ^ ro ^ ι m ^ cιi H ro oι o oθ Ln ι cιι ^ ι o cΛ Ln oι H co ι H oι ∞ o Ln o ι m ^ o m oι C ^ oι ra r ro H i ιn H oι ^ ^ o r ^ ι ri r ι CΛ ι o ^ oι ^ ^ ro θ α l H O ^ O rO M 01 VO O LO O rθ ι 01 'iJ< 00 '>il | l rθ l - rθ rθ "φ 01 0 H VO VD 01 '5}ι CO.H H OO Ol OD rO OI O OI VO Ol rO CO Ol in Ol OI CO i -O VO H '* β α) ιJl a ^|ι n ιι ri ri o ri ri o o^ o αι * ^ ^ n ^B co ψ * ^ n ι ri O H ^1 f1 Ul ιn ^ ^ ol αl O ιl Ul tø Λ lI5 ID (0 (Λ ^ <» ^1 f^ ri « ^ι β m o m ^ n N l) o o o « n M ^^ (l oo ψ o o ^ ri ω o o m ω flo ^^ o ^Λ H lrι ^J3 N rn o o <Λ ^ ol (^ n ^J3 ^ ^ ιϊl m (ll ^ (l o ol o

^ ! OO H ^ rθ 01 H ω VO rO VO Cι1 0i m Cι1 C H H ^ 03 0 01 01 ι 03 Vo σi VD I LO O OO ^ m m ι^ 01 H O VD OO H W cn ^ ι H w vo w ^ ι ^ ro ro ro ιn H ri o o oι ro Ch ji H co o ι vo co ro oi i cn m oι ω

^ uι ^ ri N rι m n N ri H θ rι cι o ιιι oι ι fi n n ^ ιι m n n * * a iιi * ri n * * * <( n ri ri o o o oι ιιι oι a) o cn o <n Q co H ro oo σ ^ ^ ^ ^ Ol Ctl O O ^ in VO LO CO O VO H O ^ t^ O O -n OO rO OO Ol LO Ol LO H VO CO H O "* L0 1 0 H H L H 0 H I ^•^^ ^J^ 0 1 VD σ ^t^,

CO H LO CO CM 01 0 ^ VO in Cι1 VD O O rθ r 01 Cι1 rO Cn σi CO LO O LO C0 01 H '* O V0 01 <ii | l 01 '* H O Ol CO VO Ln OI O CO OI H CO -O H H VD O ^ VO CO L0 l O [- in ∞ ra θ H ^ Ol rM H CΛ I O Cι1 l O H m ^ VD H ^ 00 0 01 LO VO O rO 'φ 01 LO I C0 01 01 H Ol VD VD 'φ rO CO 'ii H 'Φ LO Ol O H ^Ji VO O ro H ^ ■* Ln V0 L0 -0 in VD I l V0 V0 V0 00 01 C0 01 01 H H O CM 01 O co co vo vo -o r- co cι co co ι o o H H H H H H H H H H H H H H H H H H H H H H H H H H H H H CM CM OI OI H CM H H H H H H H H H H H H CM

ω ω ra ω ∞ o ω ra ω ∞ ω ω ω ω ffl ∞ o ω ra ∞ m m m σi m

►3 ^ H:

> > > > H H H H H H H H H H H H H H r< rf; r< <: ι ^ ι ι t <C <« <; ι O U O O O O U O O J ι- ^ ι-1 ιJ ι^ J ι-

• H Ol oi ol ro H H oi ro oi H H Ol H M H i 03 CD O rii ffl U Q W H P H N N a riJ CP O Q tH ffl O P fl s cQ O Q ω ia : m CD p Q ei m U U U U P 1-5 U U U U U U U !3 U U U U P !-3 U U U P U P S U U U P P U P S U U U U P P U P I-5 U U U U U U P S U U U P

IΛ vo o> o ri N f1 * ιn M ι- Ol O H n n * ln » ^ o rΛ O H c^ n ι* ιn ^ os (Λ O H ι^l n ^ ιn ^ ιo ιΛ O ri M ^ ^ ιl ^o ^ ι. (Λ o H M n ^ ιn vo ^ -o ι^ Ln m Lθ in ιn - Lθ i vo D vo vo vo vD o vo vD VD t^ ι c~ ^ ι ι c^ ^ c~ j oo oo co co co m o w o o ^ w vo o lo w ^D o o ^ o vo w ^o o vo -) vo vo vo o w vo vo vo vo \o ^ w w w vo ^ o w ^_^l ω o o ^ vo ^o w o ^ vo • ^ ^ ^ ^

p ε

<: : fc fc rf! rf! rf! rfj ei

ATOM 7CL6 N " TYR 91 20.415 40..821 22..672 1.,00 10,,22 APEP

ATOM 707 CA TYR 91 21, .846 40. .894 22, .380 1. ,00 10, ,21 APEP

ATOM 708 CB TYR 91 22, .426 42, .203 22, .921 1. ,00 11, ,27 APEP

ATOM 709 CG TYR 91 23, .921 42, .329 22. .730 1. ,00 13. ,72 APEP

ATOM 710 CD1 TYR 91 24, ,458 42. .653 21, .487 1. ,00 14, ,77 APEP

ATOM 711 CE1 TYR 91 25. .837 42, ,747 21, .301 1. ,00 16. ,40 APEP

ATOM 712 CD2 TYR 91 24, ,802 42, .104 23. .788 1. ,00 14, .30 APEP

ATOM 713 CE2 TYR 91 26. ,178 42, ,195 23, .614 1. ,00 14. ,06 APEP

ATOM 714 CZ TYR 91 26, .688 42, ,516 22, ,370 1. 00 18, ,00 APEP

ATOM 715 OH TYR 91 28, ,052 42, ,608 22. ,191 1. 00 18. .78 APEP

ATOM 716 C TYR 91 22, ,620 39. .714 22, ,967 1. 00 11. ,02 APEP

ATOM 717 O TYR 91 23. ,411 39. ,077 22. ,279 1. 00 11. ,79 APEP

ATOM 718 N VAL 92 22, .397 39, .432 24. ,244 1. .00 10, ,38 APEP

ATOM 719 CA VAL 92 23, .075 38, .325 24. .903 1. ,00 8, ,66 APEP

ATOM 720 CB VAL 92 22. .785 38, .319 26, .427 1. ,00 8. ,13 APEP

ATOM 721 CGI VAL 92 23, .488 37. ,142 27, .095 1. ,00 5. ,04 APEP

ATOM 722 CG2 VAL 92 23, ,267 39. ,622 27, ,046 1. ,00 6. ,97 APEP

ATOM 723 C VAL 92 22, .634 37, ,002 24, .286 1. ,00 9. ,57 APEP

ATOM 724 O VAL 92 23, ,418 36. ,063 24, ,194 1. ,00 10. ,64 APEP

ATOM 725 N ALA 93 21. ,376 36, ,933 23, ,858 1. 00 9. ,31 APEP

ATOM 726 CA ALA 93 20, ,854 35. ,722 23. ,238 1. 00 9. ,67 APEP

ATOM 727 CB ALA 93 19. ,349 35. .848 23, ,030 1. 00 8. 26 APEP

ATOM 728 C ALA 93 21. ,561 35. ,489 21. ,898 1. 00 9. 72 APEP

ATOM 729 O ALA 93 21, .954 34. ,366 21, ,581 1. 00 10. 89 APEP

ATOM 730 N GLN 94 21. ,730 36. ,565 21. ,130 1. 00 8. 57 APEP

ATOM 731 CA GLN 94 22, ,386 36. ,515 19, ,828 1. 00 6. ,19 APEP

ATOM 732 CB GLN 94 22, ,316 37. ,892 19, ,162 1. oo 7. ,13 APEP

ATOM 733 CG GLN 94 22, .606 37, .891 17, ,668 1. ,00 6. ,55 APEP

ATOM 734 CD GLN 94 21. .778 36, .875 16, .911 1. ,00 6, ,86 APEP

ATOM 735 OEl GLN 94 20, ,551 37, .018 16. ,775 1. ,00 7. .69 APEP

ATOM 736 NE2 GLN 94 22, .441 35, ,836 16, .412 1. ,00 4, .45 APEP

ATOM 737 C GLN 94 23, .843 36, .082 19, ,946 1. ,00 7, ,41 APEP

ATOM 738 O GLN 94 24, .302 35, .217 19, ,203 1. ,00 8. .55 APEP

ATOM 739 N VAL 95 24, .574 36, .699 20, .868 1. .00 7, ,81 APEP

ATOM 740 CA VAL 95 25, .971 36, ,357 21, .089 1. .00 6. ,18 APEP

ATOM 741 CB VAL 95 26, .551 37, .112 22, .327 1, ,00 8. ,06 APEP

ATOM 742 CGI VAL 95 27, .899 36, .523 22, .728 1. ,00 8. ,13 APEP

ATOM 743 CG2 VAL 95 26, .716 38. .583 22, .011 1. .00 7. ,34 APEP

ATOM 744 C VAL 95 26, .091 34, .855 21, .324 1. ,00 7, .07 APEP

ATOM 745 O VAL 95 26, .949 34, .199 20, .737 1, ,00 3. .91 APEP

ATOM 746 N TRP 96 25, .224 34, .312 22 .180 1. ,00 8, .26 APEP

ATOM 747 CA TRP 96 25 .244 32 .879 22 .494 1, .00 8 .88 APEP

ATOM 748 CB TRP 96 24 .284 32 .555 23 .650 1, .00 6 .54 APEP

ATOM 749 CG TRP 96 24 .258 31 .089 24 .030 1, .00 7 .96 APEP

ATOM 750 CD2 TRP 96 25 .390 30 .232 24 .240 1, .00 7 .64 APEP

ATOM 751 CE2 TRP 96 24 .892 28 .946 24 .549 1 .00 7 .17 APEP

ATOM 752 CE3 TRP 96 26 .778 30 .426 24 .197 1 .00 8 .39 APEP

ATOM 753 CD1 TRP 96 23 .150 30 .305 24 .217 1 .00 8 .48 APEP

ATOM 754 NE1 TRP 96 23 .524 29 .016 24 .527 1 .00 5 .50 APEP

ATOM 755 CZ2 TRP 96 25 .734 27 .859 24 .812 1 .00 6 .91 APEP

ATOM 756 CZ3 TRP 96 27 .614 29 .341 24 .459 1 .00 8 .97 APEP

ATOM 757 CH2 TRP 96 27 .087 28 .076 24 .761 1 .00 8 .90 APEP

ATOM 758 C TRP 96 24 .867 32 .033 21 .281 1 .00 8 .85 APEP

ATOM 759 O TRP 96 25 .500 31 .011 21 .007 1 .00 8 .27 APEP

ATOM 760 N ALA 97 23 .827 32 .453 20 .566 1 .00 7 .57 APEP

ATOM 761 CA ALA 97 23 .390 31 .721 19 .381 1 .00 9 .88 APEP

ATOM 762 CB ALA 97 22 .182 32 .415 18 .742 1 .00 4 .36 APEP IΛ r- vo ft ft ft Pj ft ft ft ft l-i Pi ft ft ft ft l-i pi ft lli ft ft ft ft ft ft ft ft ft ft ft ft Pi ft ft ft ft ft l-i Pi ft ft ft ft ^ r^ i^ rfl rfi ^ rfl rfl rfl rfj rfl rfj ics ^ rfj ie i^ ^ ^ i^ rfj rfj ril rfj i^ r^ rfj rfl rfj rfj rfj rf^

0 01 01 0 l O H H CO I l l CO C0 0 01 l 01 l r CM O H H 01 CO CO CO CO H CO I VD H VO '*i rO L <ii H r0 01 I 01 LO LO '^^l r0 01 ^•<*ι l O <d< Ol ^• VO rO ' O O H r^ r^ OI Ol Ol VD O VO VD H ι o o v H i ιΛ θi - tn r^ vo vo o Lθ θ i vo cι σ ι co ^ m ro ι ι H r co oo Lθ ι oo r- 01 r~ ^

H U OO CO OI OI CO CO H OI O CO CO O O O O H OI O H H cM '* ro oι ro rθ ' Vθ θi o r '«ji '* '* oi '>* ro ro H o ro ro H i -φ Εf LO rO 'φ ro ro ro l CO 01 01 α. H ^■ H H H H H H H H H H H H H H H H CM CM CM H H H H H H H H H H H H H H H H H H H H H H H H ooooooooooooooooooooooooooooooooooooooooooooooσoooooooooo ooooooooooσooooooooooooooooooooooooooσooooooooooooooooooo

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ATOM 1568 O GLY 196 11. ,649 40. ,075 32. ,596 1. 00 8, ,29 APEP

ATOM 1569 N PRO 197 12. 541 39. ,157 34. ,445 1. 00 6, ,50 APEP

ATOM 1570 CD PRO 197 11. 525 39. 698 35. ,363 1. 00 7. 41 APEP

ATOM 1571 CA PRO 197 13. 536 38. 343 35. ,153 1. 00 6. 68 APEP

ATOM 1572 CB PRO 197 13. 300 38. 688 36. ,610 1. 00 6. 91 APEP

ATOM 1573 CG PRO 197 11. .856 39, .041 36. .672 1. ,00 6, ,39 APEP

ATOM 1574 C PRO 197 13. ,266 36. ,868 34. .854 1. 00 6. ,69 APEP

ATOM 1575 O PRO 197 12. ,255 36. ,537 34, ,238 1. 00 6. ,02 APEP

ATOM 1576 N SER 198 14. ,153 35. ,975 35, ,286 1. 00 7, ,01 APEP

ATOM 1577 CA SER 198 13. ,953 34. ,557 35, ,001 1. 00 7. ,41 APEP

ATOM 1578 CB SER 198 15. .233 33. ,755 35, .303 1. ,00 4. ,71 APEP

ATOM 1579 OG SER 198 15. .558 33. .752 36. ,682 1. ,00 11, ,52 APEP

ATOM 1580 C SER 198 12. ,765 33. ,927 35, ,717 1. 00 7. ,39 APEP

ATOM 1581 O SER 198 12. ,161 34. ,528 36. ,605 1. 00 6. ,19 APEP

ATOM 1582 N GLY 199 12. ,423 32. ,716 35, ,289 1. 00 7. 86 APEP

ATOM 1583 CA GLY 199 11. ,332 31. ,977 35. ,893 1. 00 8. ,49 APEP

ATOM 1584 C GLY 199 11. ,894 30. ,622 36. ,274 1. 00 8. ,12 APEP

ATOM 1585 O GLY 199 13. 110 30. 450 36. ,306 1. 00 8. 44 APEP

ATOM 1586 N ASN 200 11. 022 29. 670 36. ,570 1. 00 9. 40 APEP

ATOM 1587 CA ASN 200 11. ,433 28. ,317 36, ,929 1. 00 11. ,14 APEP

ATOM 1588 CB ASN 200 12, ,344 27. ,742 35, .844 1. ,00 11, ,38 APEP

ATOM 1589 CG ASN 200 11. ,581 27, ,365 34. ,591 1. .00 12. ,36 APEP

ATOM 1590 OD1 ASN 200 10, ,360 27, ,478 34, ,550 1. ,00 13, ,35 APEP

ATOM 1591 ND2 ASN 200 12, ,293 26, ,919 33, ,566 1. ,00 10, ,02 APEP

ATOM 1592 C ASN 200 12, .102 28, ,177 38, .296 1. ,00 12, ,89 APEP

ATOM 1593 O ASN 200 13. .019 27, .373 38. .477 1, ,00 12, ,94 APEP

ATOM 1594 N PHE 201 11, ,632 28, ,957 39, .262 1. ,00 14, ,46 APEP

ATOM 1595 CA PHE 201 12, .157 28, .890 40, .622 1, ,00 16, .00 APEP

ATOM 1596 CB PHE 201 11, .947 30. .224 41, .339 1, ,00 14, .75 APEP

ATOM 1597 CG PHE 201 12, .805 31, .338 40, .811 1, ,00 13, .67 APEP

ATOM 1598 GDI PHE 201 12 .267 32, .311 39 .982 1. .00 13 .83 APEP

ATOM 1599 CD2 PHE 201 14 .151 31 .421 41 .157 1, .00 17 .70 APEP

ATOM 1600 CE1 PHE 201 13, .047 33, .350 39 .505 1, .00 14, .74 APEP

ATOM 1601 CE2 PHE 201 14 .948 32 .460 40 .685 1. .00 17 .45 APEP

ATOM 1602 CZ PHE 201 14 .394 33 .427 39 .857 1 .00 17 .09 APEP

ATOM 1603 C PHE 201 11 .347 27 .786 41 .304 1 .00 16 .58 APEP

ATOM 1604 O PHE 201 10 .124 27 .876 41 .385 1 .00 16 .44 APEP

ATOM 1605 N LYS 202 12 .026 26 .755 41 .797 1, .00 18 .81 APEP

ATOM 1606 CA LYS 202 11 .351 25 .617 42 .421 1 .00 21 .27 APEP

ATOM 1607 CB LYS 202 12 .386 24 .588 42 .883 1 .00 25 .00 APEP

ATOM 1608 CG LYS 202 12 .314 23 .274 42 .101 1 .00 29 .78 APEP

ATOM 1609 CD LYS 202 13 .215 22 .197 42 .698 1 .00 31 .92 APEP

ATOM 1610 CE LYS 202 12 .574 20 .816 42 .609 1 .00 32 .51 APEP

ATOM 1611 NZ LYS 202 12 .138 20 .304 43 .944 1 .00 30 .68 APEP

ATOM 1612 C LYS 202 10 .365 25 .899 43 .555 1 .00 21 .29 APEP

ATOM 1613 O LYS 202 9 .347 25 .218 43 .676 1 .00 22 .50 APEP

ATOM 1614 N ASN 203 10 .642 26 .894 44 .385 1 .00 20 .90 APEP

ATOM 1615 CA ASN 203 9 .726 27 .190 45 .485 1 .00 22 .00 APEP

ATOM 1616 CB ASN 203 10 .520 27 .657 46 .711 1 .00 23 .02 APEP

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Y 0.593

G 0.165

L 0.121

T 0.423

K 0.978

Q 0.538

E 0.264

K 0.396

Q 0.593

D 0.302

I 0.000

L 0.198

K 0.615

E 0.170

H 0.000

N 0.115

D 0.445

F 0.027

R 0.000

Q 0.198

K 0.407

I 0.000

A 0.033

R 0.956

G 0.148

L 0.593

E 0.005

T 0.610 R 0.335

G 0.110

N 0.549

P 0.363

G 0.170

P 0.440

Q 0.005

P 0.209

P 0.236

A 0.022

K 0.775

N 0.236

M 0.066

K 0.588

N 0.500

L 0.016

V 0.462

W 0.275

N 0.165

D 0.621

E 0.247

L 0.005

A 0.055

Y 0.115

V 0.005

A 0.000

Q 0.159

V 0.011

W 0.077

A 0.000

N 0.005

Q 0.027

C 0.027

Q 0.577

Y 0.687

G 0.110

H 0.549

D 0.022

T 0.429

C 0.000

R 0.203

D 0.093

V 0.044

A 0.500

K 0.824

Y 0.209

Q 0.423

V 0.011

G 0.011

Q 0.066 118 N 0.005

119 V 0.022

120 A 0.016

121 L 0.198

122 T 0.198

123 G 0.231

124 S 0.236

125 T 0.610

126 A 0.253

127 A 0.379

128 K 0.857

129 Y 0.352

130 D 0.220

131 D 0.495

132 P 0.033

133 V 0.137

134 K 0.654

135 L 0.000

136 V 0.000

137 K 0.538

138 M 0.473

139 W 0.016

140 E 0.071

141 D 0.341

142 E 0.154

143 V 0.000

144 K 0.560

145 D 0.390

146 Y 0.044

147 N 0.165

148 P 0.214

149 K 0.868

150 K 0.604

151 K 0.753

152 F 0.071

153 S 0.302

154 G 0.192

155 N 0.121

156 D 0.379

157 F 0.819

158 L 0.714

159 K 0.533

160 T 0.000

161 G 0.077

162 H 0.231

163 Y 0.000

164 T 0.000

165 Q 0.011

166 M 0.000

167 V 0.000 168 W 0.005

169 A 0.011

170 N 0.429

171 T 0.000

172 K 0.451

173 E 0.165

174 V 0.000

175 G 0.000

176 C 0.016

177 G 0.000

178 S 0.016

179 I 0.000

180 K 0.214

181 Y 0.016

182 I 0.275

183 Q 0.231

184 E 0.841

185 K 0.989

186 w 0.665

187 H 0.159

188 K 0.203

189 H 0.011

190 Y 0.000

191 L 0.000

192 V 0.000

193 C 0.000

194 N 0.000

195 Y 0.000

196 G 0.000

197 P 0.225

198 S 0.110

199 G 0.027

200 N 0.308

201 F 0.341

202 K 0.824

203 N 0.797

204 E 0.374

205 E 0.511

206 L 0.055

207 Y 0.082

208 Q 0.566

209 T 0.473

210 K 0.962

Example 10 Alignment of Ag 5s

An alignment of selected antigen 5 sequences from Vespula, Dolichovespula , Vespa, Polistes and Solenopsis (fire ants) is shown in Fig. 12. Vespula, Dolichovespula, Vespa and 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. 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 A², 585 A², 589 A², 673 A² and 1053 A², 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 A. 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 A², 496 A², 730 A², 803 A² and 1043 A², 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 π, 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

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 1/10 to 3/4 of yellow jacket antigen 5, will have fewer epitopes than the parent molecule. The CD spectral data in Figure 7 suggest that the hybrids have secondary structures closely similar, if not identical, with those of vespid antigen 5s. The inhibition data in Figures 8 and 9 with Nes 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 IgGl antibodies specific for the natural Ves v 5, six of which bound the N-terminal hybrid PVl-46. Therefore these data indicate that the hybrids contain the discontinuous B cell epitopes of Ves v 5.

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 PVl-46 are surface accessible. (See Table 7) This is in contrast to the C- terminal hybrid PVl 56-204, 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 ovotny et al., 1996, Adv Prot Chem 49:149).

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.

Our results with hybrid Ag 5 s 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.

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.

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.

All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incoφorated by reference.

Table 8. Allergens

"Clone (C) or Protein (P) data References to Appendix I

1. Marsh, D.G., and L.R. Freidhoff. 1992. ALBE, an allergen database. IUIS, Baltimore, MD, Edition 1.0.

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

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. 4. Lowenstein, H. 1980. Timothy pollen allergens. Allergy 35:188-191.

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

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.

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.

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 U, the major allergens in Ambrosia artemisiifolia (short ragweed). Int. Arch. Allergy Appl. Immunol. 96:296-304.

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.

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.

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.

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

13. EkramoddouUah, 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. 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.

15. Morgenstern, J.P., IJ. 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.

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 Fdl, the major allergen from the domestic cat. Gene 113:263-268.

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.

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.

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.

20. Ramar, 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.

21. Rogers, B.L., J.P. Morgenstern, IJ. Griffith, X.B. Yu, CM. 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. 22. Klapper, D.G., L. Goodfriend, and J.D. Capra. 1980. Amino acid sequence of ragweed allergen Ra3. Biochemistry 19:5729-5734.

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

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.

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

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

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

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.

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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), Dae g π. Allergy 48:615-623.

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

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Table 9. Selected allergens with structures available in Protein Database (PDB)

ID NO: 1A0K

Deposited: 02-Dec-1997 Exp. Method: X-ray Diffraction Resolution: 2.20 A

Title Profilin I From Arabidopsis Thaliana

Classification Cytoskeleton

Compound Mol_Id: 1; Molecule: Profilin; Chain: Null; Engineered: Recombinant Plant Protein; Biological_Unit: Monomer

ID NO: 1A9V

Deposited: 10-Apr-1998 Exp. Method: NMR, 10 Structures

Title Tertiary Structure Of The Major House Dust Mite Allergen Der P 2, NMR, 10

Structures

Classification Allergen

Compound Mol_Id: 1 ; Molecule: Mite Allergen Der P 2; Chain: Null; Engineered: Yes;

Mutation: D1S; Other_Details:DlS Mutant Made To Enhance N-Terminal Met Removal

ID NO: 1AHK

Deposited: 07-Apr- 1997 Exp. Method: NMR, Minimized Average Structure

Title Der F 2, The Major Mite Allergen From Dermatophagoides Farinae, NMR, Minimized Average Structure

Classification Allergen

Compound Mol_Id: 1; Molecule: Der F 2; Chain: Null; Synonym: Der F JJ; Engineered: Yes

ID NO: 1AHM

Deposited: 07-Aρr-1997 Exp. Method: NMR, 10 Structures

Title Der F 2, The Major Mite Allergen From Dermatophagoides Farinae, NMR, 10

Structures

Classification Allergen

Compound Mol_Id: 1 ; Molecule: Der F 2; Chain: Null; Synonym: Der F JJ; Engineered: Yes

ID NO: 1B6F

Deposited: 13-Jan-1999 Exp. Method: NMR, 23 Structures

Title Birch Pollen Allergen Bet V 1

Classification Plant Protein

Compound Mol_Id: 1 ; Molecule: Major Pollen Allergen Bet V 1-A; Chain: A; Engineered: Yes; Mutation: Yes

ID NO: 1BBG

Deposited: 24-Apr-1998 Exp. Method: NMR, Minimized Average Structure Title Ragweed Pollen Allergen From Ambrosia Trifida V, NMR, Minimized Average

Structure

Classification Allergen

Compound Mol_Id: 1; Molecule: Pollen Allergen 5; Chain: Null

ID NO: 1BJ7

Deposited: 02- Jul- 1998 Exp. Method: X-ray Diffraction Resolution: 1.80 A

Title Bovine Lipocalin Allergen Bos D 2

Classification Allergen

Compound Mol_Id: 1 ; Molecule: D 2; Chain: Null; Synonym: Dander Major Allergen Bda20, Dermal Allergen Bda20; Engineered: Yes; Biological_Unit: Monomer

ID NO: 1BMW

Deposited: 27-Jul-1998 Exp. Method: NMR, 38 Structures

Title A Fibronectin Type UI Fold In Plant Allergens: The Solution Structure Of Phi Pii

From Timothy Grass Pollen, NMR, 38 Structures

Classification Allergen

Compound Mol_Id: 1; Molecule: Pollen Allergen Phi P2; Chain: Null; Synonym: Phi P U; Engineered: Yes; Biological_Unit: Monomer

ID NO: 1BTV Deposited: 30-Jan-1997 Exp. Method: NMR, 20 Structures

Title Structure Of Bet V 1 , NMR, 20 Structures

Classification Major Birch Pollen Allergen

Compound Mol_Id: 1; Molecule: Bet V 1; Chain: Null; Engineered: Yes

ID NO: 1BV1

Deposited: 08-Jul-1997 Exp. Method: X-ray Diffraction Resolution: 2.00 A

Title Birch Pollen Allergen Bet V 1

Classification Allergen

Compound Mol_Id: 1 ; Molecule: Bet V 1 ; Chain: Null; Synonym: Major Pollen Allergen Bet V 1-A; Engineered: Yes

ID NO: 1BWH

Deposited: 24-Sep- 1998 Exp. Method: X-ray Diffraction Resolution: 1.80 A

Title The 1.8 A Stracture Of Ground Control Grown Tetragonal Hen Egg White

Lysozyme

Classification Hydrolase

Compound Mol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: Gal D IV, Allergen Gal D 4; Ec: 3.2.1.17

ID NO: 1BWI Deposited: 24-Sep- 1998 Exp. Method: X-ray Diffraction Resolution: 1.80 A

Title The 1.8 A Structure Of Microbatch Oil Drop Grown Tetragonal Hen Egg White

Lysozyme

Classification Hydrolase

Compound Mol ld: 1; Molecule: Lysozyme; Chain: A; Synonym: Gal D JN, Allergen Gal D 4; Ec: 3.2.1.17

ID NO: 1BWJ

Deposited: 18-Sep-1998 Exp. Method: X-ray Diffraction Resolution: 1.80 A

Title The 1.8 A Structure Of Microgravity Grown Tetragonal Hen Egg White

Lysozyme

Classification Hydrolase

Compound Mol ld: 1; Molecule: Lysozyme; Chain: A; Synonym: Gal D IN, Allergen Gal D 4; Ec: 3.2.1.17

ID NO: 1CQA

Deposited: 26-Jul-1996 Exp. Method: X-ray Diffraction Resolution: 2.40 A

Title Birch Pollen Profilin

Classification Contractile Protein

Compound Mol_Id: 1 ; Molecule: Profilin; Chain: Null; Engineered: Yes ID NO: 1E09

Deposited: 15-Mar-2000 Exp. Method: NMR, 22 Structures

Title Solution Structure Of The Major Cherry Allergen Pru Av 1

Classification Allergen

Compound Mol_Id: 1; Molecule: Pru Av 1; Chain: A; Engineered: Yes

ID NO: 1EW3

Deposited: 21-Aρr-2000 Exp. Method: X-ray Diffraction Resolution: 2.30 A

Title Crystal Structure Of The Major Horse Allergen Equ C 1

Classification Allergen

Compound Mol_Id: 1 ; Molecule: Allergen Equ C 1 ; Chain: A; Engineered: Yes

ID NO: 1F2K

Deposited: 26-May-2000 Exp. Method: X-ray Diffraction Resolution: 2.30 A

Title Crystal Structure Of Acanthamoeba Castellanii Profilin IJ, Cubic Crystal Form

Classification Structural Protein

Compound Mol_Id: 1; Molecule: Profilin II; Chain: A, B; Engineered: Yes

ID NO: 1FCQ

Deposited: 19-Jul-2000 Exp. Method: X-ray Diffraction Resolution: 1.60 A Title Crystal Structure (Monoclinic) Of Bee Venom Hyaluronidase

Classification Hydrolase

Compound Mol_Id: 1; Molecule: Hyaluronoglucosamimdase; Chain: A; Synonym: Hyaluronidase, Api M JJ; Ec: 3.2.1.35; Engineered: Yes

ID NO: 1FCU

Deposited: 19-Jul-2000 Exp. Method: X-ray Diffraction Resolution: 2.10 A

Title Crystal Structure (Trigonal) Of Bee Venom Hyaluronidase

Classification Hydrolase

Compound Mol ld: 1; Molecule: Hyaluronoglucosamimdase; Chain: A; Synonym: Hyaluronidase, Api M JJ; Ec: 3.2.1.35; Engineered: Yes

ID NO: 1FCV

Deposited: 19-Jul-2000 Exp. Method: X-ray Diffraction Resolution: 2.65 A

Title Crystal Structure Of Bee Venom Hyaluronidase In Complex With Hyaluronic

Acid Tetramer

Classification Hydrolase

Compound Mol_Id: 1; Molecule: Hyaluronoglucosamimdase; Chain: A; Synonym: Hyaluronidase, Api M II; Ec: 3.2.1.35; Engineered: Yes

ID NO: 1FLQ Deposited: 15-Aug-2000 Exp. Method: X-ray Diffraction Resolution: 1.80 A

Title Hen Egg White Lysozyme Mutant With Alanine Substituted For Glycine

Classification Hydrolase

Compound Mol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: 1,4~N-Acetylmuramidase C, Allergen Gal D 4, Gal D TV; Ec: 3.2.1.17; Engineered: Yes; Mutation: Yes

ID NO: 1FLU

Deposited: 15-Aug-2000 Exp. Method: X-ray Diffraction Resolution: 1.78 A

Title Hen Egg White Lysozyme Mutant With Alanine Substituted For Glycine

Classification Hydrolase

Compound Mol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: 1,4~N-Acetylmuramidase C, Allergen Gal D 4, Gal D IV; Ec: 3.2.1.17; Engineered: Yes; Mutation: Yes

ID NO: 1FLW

Deposited: 15-Aug-2000 Exp. Method: X-ray Diffraction Resolution: 1.81 A

Title Hen Egg White Lysozyme Mutant With Alanine Substituted For Glycine

Classification Hydrolase

ID NO: 1FLY Deposited: 15-Aug-2000 Exp. Method: X-ray Diffraction Resolution: 1.83 A

Title Hen Egg White Lysozyme Mutant With Alanine Substituted For Glycine

Classification Hydrolase

Compound Mol ld: 1; Molecule: Lysozyme; Chain: A; Synonym: 1,4~N-Acetylmuramidase C, Allergen Gal D 4, Gal D IV; Ec: 3.2.1.17; Engineered: Yes; Mutation: Yes

ID NO: 1FN5

Deposited: 2 l-Aug-2000 Exp. Method: X-ray Diffraction Resolution: 1.78 A

Title Hen Egg White Lysozyme Mutant With Alanine Substituted For Glycine

Classification Hydrolase

Compound Mol_Id: 1; Molecule: Lysozyme; Chain: A; Synonym: 1,4~N-Acetylmuramidase C, Allergen Gal D 4, Gal D JN; Ec: 3.2.1.17; Engineered: Yes; Mutation: Yes

ID NO: 1FSK

Deposited: 1 l-Sep-2000 Exp. Method: X-ray Diffraction Resolution: 2.90 A

Title Complex Formation Between A Fab Fragment Of A Monoclonal IgG Antibody and The Major Allergen From Birch Pollen Bet V 1

Classification Immune System

Compound Mol ld: 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: Bvl6 Fab-Fragment, Moρc21 Coding Sequence; Engineered: Yes Mol_Id: 3; Molecule: Antibody Heavy Chain Fab; Chain: C, F, I, L; Synonym: Heavy Chain Of The Monoclonal Antibody Mst2; Engineered: Yes

ID NO: 1G5U

Deposited: 02-Nov-2000 Exp. Method: X-ray Diffraction Resolution: 3.10 A

Title Latex Profilin Hevb8

Classification Allergen

Compound Mol_Id: 1; Molecule: Profilin; Chain: A, B; Engineered: Yes

ID NO: 1H6M

Deposited: 19-Jun-2001 Exp. Method: X-ray Diffraction Resolution: 1.64 A

Title Covalent Glycosyl-Enzyme Intermediate Of Hen Egg White Lysozyme

Classification Hydrolase (O-Glycosyl)

Compound Mol_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 Intermediate

ID NO: 1 JTI

Deposited: 21-Aug-2001 Exp. Method: X-ray Diffraction Resolution: 2.30 A

Title Loop-Inserted Structure OfPl-Pl' Cleaved Ovalbumin Mutant R339T

Classification Allergen Compound Mol_Id: 1; Molecule: Ovalbumin; Chain: A, B; Engineered: Yes; Mutation: Yes

ID NO: 1JTT

Deposited: 22-Aug-2001 Exp. Method: X-ray Diffraction Resolution: 2.10 A

Title Degenerate Interfaces In Antigen- Antibody Complexes

Classification Immune System, Lysozyme

Compound Mol_Id: 1; Molecule: Nh Single-Domain Antibody; Chain: A; Fragment: Nh

Domain Fragment; Engineered: Yes Mol_Id: 2; Molecule: Lysozyme; Chain: L; Fragment: Enzyme; Synonym: 1,4~Ν-Acetylmuramidase C, Allergen Gal D IV; Ec: 3.2.1.17

ID NO: 1K0K

Deposited: 19-Sep-2001 Exp. Method: X-ray Diffraction Resolution: 2.35 A

Title Yeast Profilin, Cubic Crystal Form

Classification Contractile Protein

Compound Mol_Id: 1; Molecule: Profilin; Chain: A; Engineered: Yes

ID NO: 1KKC

Deposited: 07-Dec-2001 Exp. Method: X-ray Diffraction Resolution: 2.00 A

Title Crystal Structure Of Aspergillus Fumigatus Mnsod

Classification Oxidoreductase Compound Mol ld: 1; Molecule: Manganese Superoxide Dismutase; Chain: A, B, X, Y; Synonym: Mnsod; Ec: 1.15.1.1; Engineered: Yes

ID NO: 1KUR

Deposited: 22-Jan-2002 Exp. Method: Theoretical Model

Title Theoretical Model Of The Allergen Jun A 3 From Mountain Cedar Pollen

Classification Allergen

Compound Mol_Id: 1; Molecule: Allergen Jun A 3; Chain: A; Synonym: Pathogenesis-Related Protein

ID NO: 1PLM

Deposited: 09-Jan-1998 Exp. Method: Theoretical Model

Title Arabidopsis Profilin 1 Complexed With Poly-L-Proline, Theoretical Model

Classification Complex (Protein/Peptide)

Compound Mol_Id: 1; Molecule: Profilin 1; Chain: A; Engineered: Yes Mol_Id: 2; Molecule: Poly-L-Proline; Chain: B; Engineered: Yes

ID NO: 1PRQ

Deposited: 18-Aug-1997 Exp. Method: X-ray Diffraction Resolution: 2.50 A

Title Acanthamoeba Castellanii Profilin la

Classification Contractile Protein Compound Mol_Id: 1; Molecule: Profilin la; Chain: Null; Engineered: Yes

ID NO: 1QMR

Deposited: 06-Oct-1999 Exp. Method: X-ray Diffraction Resolution: 2.15 A

Title Birch Pollen Allergen Bet V 1 Mutant N28T, K32Q, E45S, P108G

Classification Allergen

Compound Mol_Id: 1 ; Molecule: Major Pollen Allergen Bet V 1-A; Chain: A; Synonym: Bet V 1; Engineered: Yes; Mutation: Yes

ID NO: 1QNX

Deposited: 25-Oct- 1999 Exp. Method: X-ray Diffraction Resolution: 1.90 A

Title Ves V 5, An Allergen From Vespula Vulgaris Venom

Classification Allergen

Compound Mol_Id: 1; Molecule: Ves V 5; Chain: A; Synonym: Antigen 5; Engineered: Yes

ID NO: 1WHO

Deposited: 04-Aρr-1997 Exp. Method: X-ray Diffraction Resolution: 1.90 A

Title Allergen Phi P 2

Classification Allergen

Compound Moljd: 1 ; Molecule: Allergen Phi P 2; Chain: Null; Synonym: Phi P JJ; Engineered: Yes

ID NO: 1 HP

Deposited: 04-Aρr-1997 Exp. Method: X-ray Diffraction Resolution: 3.00 A

Title Allergen Phi P 2

Classification Allergen

ID NO: 2BBG

Deposited: 24-Aρr-1998 Exp. Method: NMR, 30 Structures

Title Ragweed Pollen Allergen From Ambrosia Trifida V, NMR, 30 Structures

Classification Allergen

Compound Moljd: 1; Molecule: Pollen Allergen 5; Chain: Null

ID NO: 3BBG

Deposited: 24-Apr-1998 Exp. Method: NMR, 2 Structures

Title Multi-Conformer Stracture Of Ragweed Pollen Allergen From Ambrosia Trifida

V, NMR, 2 Structures

Classification Allergen Compound Moljd: 1; Molecule: Pollen Allergen 5; Chain: Null

ID NO: 3NUL

Deposited: 27-Nov- 1996 Exp. Method: X-ray Diffraction Resolution: 1.60 A

Title Profilin I From Arabidopsis Thaliana

Classification Actin-Binding Protein

Compound Moljd: 1; Molecule: Profilin I; Chain: Null; Engineered: Selenomethionyl Protein

Claims

claim: 1. An allergen hybrid protein having reduced allergenicity but retaining immunogenicity, comprising a peptide epitope sequence of an allergen protein and a scaffold protein that is structurally homologous to the allergen protein, wherein the hybrid protein has a native conformation and the peptide epitope sequence is present in a surface accessible region of the hybrid protein conesponding to its position in the allergen protein.

2. The hybrid protein of claim 1 wherein the peptide epitope sequence is in a loop or corner region of the hybrid protein.

3. The hybrid protein of claim 1 wherein the scaffold protein has at least 50 percent sequence identity to the allergen from which the peptide epitope sequence is derived.

4. The hybrid protein of claim 1 wherein the scaffold protein does not have more than 70 percent sequence identity to the allergen protein from which the peptide epitope sequence is derived.

5. The hybrid protein of claim 1 wherein the peptide epitope sequence is about 6 to about 55 amino acids in length.

6. The hybrid protein of claim 5 wherein the peptide epitope sequence is about 6 to about 45 amino acids in length.

7. The hybrid protein of claim 6 wherein the peptide epitope sequence is about 6 to about 35 amino acids in length.

8. The hybrid protein of claim 7 wherein the peptide epitope sequence is about 6 to about 25 amino acids in length.

9. The hybrid protein of claim 8 wherein the peptide epitope sequence is about 6 to about 15 amino acids in length.

10. The hybrid protein of claim 1 further comprising a signal peptide.

11. The hybrid protein of claim 1 further comprising a protease processing site.

12. The hybrid protein of claim 1 which is a hybrid vespid venom allergen protein.

13. The hybrid protein of claim 12, which is a hybrid vespid venom antigen 5 protein.

14. The hybrid protein of claim 13 wherein the peptide epitope sequence is from the genus Vespula and the scaffold protein is from the genus Polistes.

15. The hybrid protein of claim 14 wherein the peptide epitope sequence is from the species vulgaris.

16. The hybrid protein of claim 14 wherein the scaffold protein is from the species annularis.

17. The hybrid protein of claim 13 wherein the peptide antigen comprises a sequence selected from the group consisting of

NNYCKJKC (SEQ ID: 1); NNYCKIKCLKGGVHTACK (SEQ ID: 2); NNYCKJ CLKGGVHTACKYGSLKP (SEQ ID: 3); NNYCKTJ CLKGGVHTACKYGSLKPNCGNKWN (SEQ ID: 4); ΝΝYCKIKCLKGGVHTACKYGSLKPΝCGΝKVWSYGLTKQ (SEQ ID: 5); ΝΝYCKIKCLKGGVHTACKYGSLKPΝCGΝKVWSYGLTKQEKQDILK (SEQ ID: 6); QVGQΝVALTGSTAAKYDDPVKLVKMWEDEVKDYΝPKKKFSGΝDFL KTG (SEQ JJ ΝO: 7); IJNTQMVWANTKEVGCGSIKYIQEKWHPIYLVCNYGPSGNFKNEEL YQTK (SEQ ID NO: 8) LKPNCGNKVW (SEQ ID NO: 9); LTGSTAAKYDD (SEQ ID NO: 10); PKKKFSGND (SEQ ID NO: 11) IQJXWHK (SEQ JD NO: 12); and FKNEELYQTK (SEQ ID NO: 13); NNYCKJKCLKGGVHTACKYGSLKPNCGNKVNVSYGLTKQEKQDILK EHΝD (SEQ ID NO: 93); NNYCKJKCLKGGVHTACKYGSLKPNCGNKWVSYGLTKQEKQDILK EHNDFRQKIAR (SEQ ID NO: 94); NNYCJilKCLKGGVHTACKYGSLKPNCGNKNVVSYGLTKQEKQDILK EHNDFRQKI-ARGLETRGNPGPQPPAKNlVfKN (SEQ ID NO: 95).

18. The hybrid protein of claim 1 wherein the peptide epitope sequence comprises a conservative amino acid change.

19. The hybrid protein of claim 18 wherein the variant peptide is characterized as reducing antibody binding to the peptide epitope sequence by at least 50- percent in an in vitro assay, wherein the variant is present in the assay at a concentration less than 10-fold greater than the peptide epitope sequence, and the assay measures binding of the peptide epitope sequence to an antibody directed against a polypeptide comprising the peptide epitope sequence.

20. A nucleic acid encoding the allergen hybrid protein of any one of claims 1-19.

21. A method for preparing a nucleic acid that encodes an allergen hybrid protein; which 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, wherein the nucleotide sequence encoding the peptide epitope sequence is in-frame with the nucleotide sequence encoding the scaffold protein and is 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.

22. The method according to claim 21 , wherein the nucleotide sequence encoding the scaffold protein is mutated to introduce the nucleotide sequence encoding the peptide epitope sequence.

23. The method according to claim 21 , wherein 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.

24. A nucleic acid prepared according to the method of claim 21.

25. An expression vector comprising the isolated nucleic acid of claim 20 operationally associated with a promoter.

26. A method for producing an allergen hybrid protein with reduced allergenicity but retaining immunogenicity, which method comprises culturing a cell transformed with the expression vector of claim 25 so that the hybrid allergen is produced by the cell.

27. The method of claim 26, which further comprises recovering the hybrid allergen from the culture, the cell, or both.

28. A method for treating an allergic condition, which method comprises administering a therapeutically effective amount of the hybrid protein of claim 1 or the expression vector of claim 25 to a patient who is allergic to the allergen protein or the scaffold protein, or both.

29. The method of claim 28, wherein the hybrid protein or expression vector is administered orally, pulmonarily, nasally, topically or parenterilly.

30. A pharmaceutical composition comprising the hybrid protein of claim 1 or the expression vector of claim 25 and a pharmaceutically acceptable diluent or carrier.

31. A method of designing a hybrid allergen of reduced allergenicity but retaining immunogenicity, which method comprises (a) identifying a solvent exposed surface of an allergen; (b) identifying a protein that is stracturally homologous to the allergen; and (c) modifying sequence of the protein that is structurally homologous to the allergen to incoφorate a peptide sequence from the solvent exposed surface of the allergen.

32. The method of claim 31 wherein said solvent exposed surface is identified by a physical means.

33. The method of claim 32 wherein said physical means is x-ray crystallography.

34. The method of claim 31 wherein said solvent exposed surface is identified by comparing the amino acid sequence of the allergen to the amino acid sequence of a structurally homologous protein of known three-dimensional structure.

35. The method of claim 31 , wherein the solvent exposed surface comprises a loop or a corner region.