The research leading to the present invention was supported by United States Public Health Service Grant No AI-17021 The government may have certain rights in the invention

FIELD OF THE INVENTION

The present invention is directed to immunogenic peptides from vespid antigen 5 These immunogenic peptides can be used in immunotherapy for vespid venom allergic individuals The present invention is thus directed to T cell epitopes of vespid antigen 5 that can anergize T cell responses in sensitive individuals

BACKGROUND OF THE INVENTION

Biochemical Aspects of Insect Venom Allergens

Insect sting allergy to bees and vespids is of common occurrence The vespids include hornets, yellowjackets and wasps [Golden, et al , Am Med Assoc , 262 240 (1989)] 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, Ann Allergy , 52 276 (1984)]

Indeed, venom allergens from insects of the Hymenoptera order have been extensively studied. These insects are bees, vespids and fire ants The bees include honey bees (Apis meliferά) and bumble bees (Bombus pennsylvamcua) The vespids include hornets (Dohchovespula spp , Vespa crabo), yellow jackets (Vespula spp ), and paper wasps (Polistes spp ) In Table 1 are listed the venom allergens from these insects with known primary structures

Paper wasp, Pohstes annulans

Pol 1 5 antigen 5 23 kd +

Yellow jacket, Vepula vulgans

Ves v 1 phospholipase A, 34 kd + Ves v 2 hyaluronidase 38 kd + Ves v 5 ⁵ antigen 5 23 kd +

Footnotes 1 Allergen names are designated according to an accepted nomenclature system

[King et al , WHO Bulletin, 72 797 (1994)]

2 Several allergens are glycoproteins, and the molecular size given refers only to the protein portion

3 + and - signs refer to the availability of recombinant proteins 4 Sequence of antigen 5 from S nchteria is known [Smith & Hoffman, J Allerg

Clin Imunol , 89 293 (1992)] 5 Sequences of antigen 5s from several other vespids are known, D arenaria [Lu et al , J Immunol 150 2823 (1993)], P exclamans and P fuscatus, and V flavopilosa, V germamca, V maculifrons, V pennsylvamca, V spamosa and V vidua [Hoffman et al , Int Archs Allergy App Immunol 84 24 (1987)]

There are many species of hornets (genus Dolichovespula), yellowjackets (genus Vespula) and wasp (genus Polistes) in North America [Akre, et al. , "Yellowjackets of America North of Mexico," Agriculture Handbook No. 552, US Department of Agriculture (1980)]. The vespids have similar venom compositions [King, et al. , Biochemistry , 17:5165 (1978); King, et al., Mol. Immunol. 20:297 (1983); King, et al.. Arch. Biochem. Biophys. 230: 1 (1984); King, et al., J. Allergy and Clin. Immunol. , 75:621 (1985); King, J. Allergy Clin. Immunol. , 79: 113 (1987); Hoffman, /. Allergy and Clin. Immunol. , 75:611 (1985)]. Their venom each contains three major venom allergens, phospholipase (37 kD), hyaluronidase (43 kD) and antigen 5 (23 kD) of as yet unknown biologic function. Homologous venom allergens from hornets, yellow jackets, and paper wasps have high degrees of sequence identity ranging form about 70% for antigen 5s to about 90% for hyaluronidases [Lu et al., J. Immunol. , 150:2823 (1993)].

Antigen 5s from several species each of hornets, yellowjackets and paper wasps have been cloned and/or sequenced [Fang et al. , Proc. Natl. Acad. Sci., USA, 85:895-899 (1988); Lu et al., supra; Hoffman, J. Allergy Clin. Immunol. , 92:707-716 (1993)]. For phospholipases and hyaluronidases only those from hornets and yellowjackets have been cloned and/or sequenced [Soladatova et al. , FEBS Letters, 320: 145-149 (1993); Lu et al. , J. Biol. Chem. , 270:4457-4465 (1995); King et al.. J. Allergy Clin. Immunol. , (1996); Hoffman, Int. Arch. Allergy Immunol. , 104: 184-190 (1994)]. One common feature of these venom proteins is their varying extent of sequence homology with mammalian proteins.

White faced hornet (Dolichovespula maculata) has three forms of antigen 5. Two of these forms, Doi m 5.01 and 5.02, differ in 23 % of their sequences, and they are antigenically cross reactive at both B and T cell levels [Fang et al. , Proc. Natl. Acad. Sci., USA, 85:895-899 (1988); Lu et al., J. Immunol. , 150:2823-2830 (1993)]. The studies described here were made with Doi m 5.02, also referred to as hornet Ag5 form 2. The amino acid sequences of Doi m 5.01 and 5.02, as well as those of the homologous antigen 5s from yellow hornet (Dolichovespula arenaria), two species of yellowjacket (Vespula maculifrons and vulgaris) and two species of papers wasps (Polistes annularis and exclamans) are given in Figure 1.

Fire ant venom contains four allergens. They are antigen 5, phospholipase A,, and Sol i 2 and 4. Fire ant antigen 5 has about 50% sequence identity with vespid antigen 5 [Hoffman, J. Allergy Clin. Immunol. , 91:71 (1995)]. Only partial sequence data is available for fire ant phospholipase, and it shows sequence identity with vespid phospholipase A, [Hoffman, J. Allergy Clin. Immunol. , 95:372 (1995)].

Bumble bee venom has two allergens of known sequences; phospholipase A ₂ and protease. But honey bee venom has four allergens of know sequences; acid phosphatase, phospholipase A ₂ , hyaluronidase and a cytolytic peptide melittin. The two bee venom phospholipase A ₂ have extensive sequence identity and they are not-related to vespid phospholipase A, [Hoffman, "Hymenoptera Venom Proteins" in National Toxins, R.B. Singh (ed ), Plenum Publishing 6 (1996)]. Honey bee venom hyaluronidase has about 55 % sequence identity with the homologous vespid hyaluronidases.

In addition to the insect venom allergens described above, the complete amino acid sequence of several major allergens from different grass [Perez, et al. , J. Biol. Chem. , 265: 16210 (1990); Ansari, et al., Biochemistry, 26:8665 (1989); Silvanovich, et al., . Biol. Chem. , 266: 1204 (1991)], tree pollen [Breiteneder, EMBO J. , 8: 1935(1989); Valenta, et al. , Science, 253:557 (1991)], weed pollen [Rafnar, et al. , J. Biol. Chem. , 266: 1229 (1991); Griffith, et al. , Int. Arch. Allergy Appl. Immunol. , 96:296 (1991)], mites [Chua, et al. , J. Exp. Med. , 167: 175 (1988)], cat dander [Griffith, et al., Gene. , 113:263 (1992)], and mold [Aruda. et al. , J. Exp. Med. , 172: 1529 (1990); Han, et al. , J. Allergy Clin. Immunol. , 87:327 (1991)] have been reported in the past few years. 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.

T and B Cell Epitopes 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, Current Opinion in Immunol, 4:63 (1992)]. As MHC molecules are highly polymorphic in individuals, they have different specificity of binding antigenic peptides [Rothbard and Gefter, Ann. Rev. Immunol. , 9:527 (1991)]. 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, FASEB J. , 5:171 (1991)] and apparently in humans [Wierenga, et al. , J. Immunol. , 144:4651 (1990); Parronchi, et al. , Proc. Natl. Acad. Sci. USA. , 88:4538 (1991)] the T helper cells can be divided into different types on the basis of their patterns of lymphokine production. Primarily, T helper cells divide into two groups: TH1 cells producing IL-2 and IFN-γ, and TH2 cells producing IL-4 and IL-5. These lymphokines in turn influence the antigen-activated B cells to differentiate and proliferate into plasma cells secreting Abs of different isotypes. IL-4 is one lymphokine known to influence IgE synthesis [Finkelman, et al. , Ann. Rev. Immunol. , 8:303 (1990)].

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. , Ann. Rev. Immunol. , 2:67 (1984)]. 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 [A it, et al., Science, 233:747 (1986)], but peptide mapping suggests that less than about 8 residues contribute significantly to the binding process of a linear epitope [Appel, et al. , J. Immunol. , 144:976 (1990)].

Allergens, like other protein antigens, can have both types of B cell epitopes or only one. For example, vespid antigen 5s have both types [King et al.. J. Immunol. , 154:577 (1995)]. Bee venom melittin appears to have only one B cell epitope of linear type [King, et al., J. Immunol. , 133:2668 (1984)].

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 of unknown specificity [Unanue,

Curr Op Immunol , 4 63 (1992)] 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 ammo acid residues [Cf Rudensky et al , Nature, 353 622 (1991)] 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 , Immunol Rev , 98 53 ( 1987), O'Hehir et al , Ann Rev Immunol , 9 67 (1991)]

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- nkmg of cell-bound IgE by allergens leads to release of histamine, leukotπenes and other chemical mediators that cause the allergic symptoms IgE is one of the different isotypes of lmmunoglobulins 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 isotypes switch event of B cells, the T cell epitopes of several allergens have been mapped [Cf O'Hehir et al , supra] These allergens include ragweed Amb a III, rye grass Lol p I, cat Fel d I, mouse urine Mus m I, midge Chi 1 1, bee venom phospholipase A ₂ [Dhillon, et al , J Allergy Clin Immunol , 90 42 (1992)] melittin [Fehlner, et al . J Immunol , 146 799 (1991)]. and hornet antigen 5 [King et al , J Allergy Clin Immunol , 91 283 (1993)] 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 , Immunol Today , 12 193 (1991). Basten, et al , Immunol Rev , 122 5 (1991)] 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 multi-valent antigens which can cross-link cell surface Ig receptors [DeFranco, Ann Rev Cell Biol , 3 143 (1987)], 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, Science, 248 1349 (1990), Jenkins and Miller, FASEB J , 6 2428 (1992)] Interaction of TCR with peptide-MHC complex in absence of co-stimulating signals can lead to T cell anergy

The molecular mechanism of B or T cell anergy is not yet understood [Cf Schwartz,

1990, supra, Jenkins and Miller, 1992, supra, Ales-Martinez, et al , Immunol Today, 12 201 (1991)] In vitro studies with T cell clones reveals that occupancy of TCR by artificial peptide-MHC complex in absence of co-stimulating signals leads to altered intracellular signal transduction and/or repressor gene activation which can prevent lymphokine transcription

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

One way to treat allergic diseases is by immunotherapy which involves repeated subcutaneous injections of the offending allergen(s) into patients The amounts of allergens which can be injected are limited by the danger of unwanted systemic allergic reaction in patients For most patients following immunotherapy, their allergen-specific IgE levels initially rise followed with gradual decrease of their allergen-specific IgE levels, and there is also down-regulation of allergen-specific T cell responses [P S Norman, Current Op Immunol , 5 968 (1993)]

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, in "Bronchial Asthma," edited by E B Weiss and M Stein, Little Brown, Boston, pp 43-49 (1993), R E O'Hehir et al ,

1991 , supra]

Three reports have appeared recently 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. , Proc. Natl. Acad. Sci. U.S.A.. 90:7608-12 (1993)]. 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. , J. Exp. Med. , 178: 1783-1788 (1993)]. The third reports that peptides containing T cell epitopes of bee venom phospholipase A _: were used successfully in immunotherapy to treat patients with bee venom allergy [Miiller et al., J. Allergy Clin. Immunol. 97:426 (1996)].

Since an MHC class II molecule of any one haplotype can bind a wide range of peptides in its binding groove, it may be possible to modulate T cell response by inhibition of allergen-derived T cell epitope binding to MHC molecules with other peptides. For example, a mouse lysozyme peptide which is not immunogenic by itself in H-2 mice inhibits T cell response to hen egg white lysozyme [Adorini and Nagy, Immunol. Today. , 11:21 (1990)]. Another example is the in vitro inhibition of T cell response to a mite allergen by an influenza HA peptide [O'Hehir et al. , J. Allergy Clin. Immunol. , 87: 1 120 (1991)].

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

It has been reported that subcutaneous or intranasal pretreatment of mice with T cell epitopes peptides of the major cat allergen Fel d 1 [Briner et al. , Proc. Natl. Acad. Sci. USA, 90:7608-7612 (1993)] or the major mite allergen Der p 1 [Hoyne et al. , J. Exp. Med., 178: 1783-1788 (1993)] lead to T cell anergy and that reduced antibody responses are observed on subsequent immunization with the allergen. T cell epitope peptides of Fel d 1 studied in the murine system are currently being evaluated for immunotherapy of patients [Norman et al. , J. Allergy Clin. Immunol. , 95:259 (1995)]. Published data

indicate that the T cell epitope regions of Der p 1 detected in the muπne system overlap those found in humans [O'Hehir et al , Europ J Clin Invest , 23 763 (1993)] On the basis of findings with Fel d 1 and Der p 1 , it is reasonable to conclude that the T cell epitope data of white faced hornet Ag5 obtained in mice as described herein will be applicable for studies in humans

There remains a need in the art for the identification of T cell epitopes of vespid venom allergens, particularly antigen 5, for immunotherapy of venom allergy

There is also a need in the art to use peptides having T cell epitopes of vespid venom allergens to study induction of tolerance in mice and induction of tolerance in humans

There is a further need to test whether a modified peptide inhibits allergen T cell epitope binding to MHC class II molecule, or induces T cell anergy, or both

These and other needs in the art are satisfied by the present invention

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 the sequences of immunodominant peptides of vespid venom antigen 5, and corresponding peptides from other antigens, such as fire ant Sol I 3 An immunodominant peptide is one that contains a T cell epitope of the antigen, such that T cells immunized with the antigen will be stimulated when contacted with the peptide Such peptides of the invention are preferably l munomodulatory peptides as well, in that they induce T cell anergy when administered to a subject, or otherwise affect the immune response of the subject

In yet another embodiment, the present invention provides a pharmaceutical composition effective for the treatment of a vespid venom allergen-specific allergic condition comprising a polypeptide of the invention that has an lmmunomodulatory portion of a T cell epitope of a vespid venom antigen 5 More particularly, the invention provides

pharmaceutical compositions comprising such polypeptides, including different isoforms, of a vespid venom antigen 5, for example, Dolichovespula maculata, Vespula vulgans, Vespula maculifrons, Dolichovespula arenaria, Polistes annularis, and Polistes exclamans

In yet still another embodiment, the present invention provides a method for treating a vespid venom allergen-specific condition comprising administering a therapeutically effective dose of a pharmaceutical composition of the invention

In its broadest aspect, the present invention is directed to a peptide characterized by having between 8 (the generally recognized minimum number of am o acids for an antigenic T cell epitope) and 35 ammo acid residues of vespid venom antigen 5, and being antigenic for T cell proliferation in a mouse immunized with a vespid venom antigen 5, which mouse is a strain selected from the group consisting or BALB/c, ASW/Sn, C3H/He, and P/J More particularly, a peptide of the invention corresponds to a fragment of white face hornet antigen 5, form 2, selected from the group consisting of NNYCKIKCRKGIHTLCKFGT (SEQ ID NO 8),

GIHTLCKFGTSMKPNCGRNV (SEQ ID NO 9),

KNEILKRHNDFRQNVAKGLE (SEQ ID NO 12),

FRQNVAKGLETRGKPGPQPP (SEQ ID NO 13), TRGKPGPQPPAKNMNVLVWN (SEQ ID NO 14),

DELAKIAQTWANQCDFNHDD (SEQ ID NO 16),

CRNTAKYQVGQNIAISSTTA (SEQ ID NO 18)

QNIAISSTTATQFDRPSKLI (SEQ ID NO 19),

TQFDRPSKLIKQWEDEVTEF (SEQ ID NO 20), KQWEDEVTEFNYKVGLQNSN (SEQ ID NO 21),

NYKVGLQNSNFRKVGHYTQM (SEQ ID NO 22),

FRKVGHYTQMVWGKT (SEQ ID NO 23),

KEIGCGSIKYIEDNWYTHYL (SEQ ID NO 25). and

IEDNWYTHYLVCNYGPGGND (SEQ ID NO 26) Such peptides may be from the specific antigen, white face hornet antigen 5, form 2, or may be from the corresponding (homologous) segments of other vespid venom antigens, such as but not limited to Dolichovespula maculata, Vespula vulgans, Vespula maculifrons, Dolichovespula arenaria, and Polistes exclamans Other sources of antigen 5 of the invention include Vespa crabo (European hornet), V flavopilosa (yellow jacket), V

germanica (yellowjacket), V. peήnsylvanica (yellowjacket), V. squamosa (yellowjacket), V. vidua (yellowjacket), and P fuscatus (paperwasp), or fire ant allergen Sol i 3, which shares sequence similarity with (and possibility is homologous to) vespid venom antigen 5s. Such corresponding peptides from various species are referred to herein as homologues, as they represent variants from the corresponding, i.e. , homologous, alleles of the antigen 5 gene from different vespid species. The term allelic variant includes, as well, single amino acid variations in antigen 5s from the same species, including different isoforms (such as the three isoforms found for white face hornet antigen 5), and mutations .

Preferably, the peptide corresponds to a fragment of white face hornet antigen 5, form 2, or a homologue thereof, selected from the group consisting of:

NNYCKIKCRKGIHTLCKFGT (SEQ ID NO:8);

GIHTLCKFGTSMKPNCGRNV (SEQ ID NO:9); KNEILKRHNDFRQNVAKGLE (SEQ ID NO: 12);

FRQNVAKGLETRGKPGPQPP (SEQ ID NO: 13);

DELAKIAQTWANQCDFNHDD (SEQ ID NO : 16) ;

NYKVGLQNSNFRKVGHYTQM (SEQ ID NO:22);

FRKVGHYTQMVWGKT (SEQ ID NO:23); and IEDNWYTHYLVCNYGPGGND (SEQ ID NO:26).

This group of peptides showed significant T cell stimulation of antigen 5-immunized T cells in more than one species.

As pointed out above, the present invention advantageously provides not only the immunodominant T cell epitopes for white face hornet antigen 5, but corresponding epitopes from antigen 5s from other vespid species, particularly subfamilies Vespinae and Polistinae, more particularly the genera Vespa, Vespula, Dolichovespula, and Polistes. In addition, the invention provides for consensus polypeptides, which incorporate the same amino acid residues at conserved positions, and any amino acid residue or amino acid residues having similar properties at divergent positions. In other words, the present invention contemplates immunodominant peptides that incorporate polymorphisms found among the antigen 5 homologues from different species.

In a specific embodiment, the peptide has the ammo acid sequence X^YCKIX^X^GXsX.HTX^XAG (SEQ ID NO 31), wherein X, is a neutral ammo acid residue, X ₂ is a basic amino acid residue or deleted, X ₃ is any amino acid residue, X ₄ is a polar ammo acid, X, is glycine or deleted, X _ft is any ammo acid residue, X ₇ is an amino acid residue with an aliphatic side chain, X ₈ is a polar amino acid residue, and X, is an amino acid residue with an aromatic side chain In a more specific embodiment, X, is valine or asparagine, X ₂ is lysine, arginine, or deleted, X, is proline, argmine, serine, or leucine, X ₄ is arginine, lysine, or serine, X ₆ isoleucine, leucine, threonine, or valine, X ₇ is alanine, valine, isoleucine, or leucine, X ₈ is lysine, arginine, glutamine, or asparagine, or X, is phenylalanine, tryptophan, or tyrosine, or any combination of the foregoing

In another specific embodiment, the peptide has the amino acid sequence GXjX ₆ HTX ₇ CX ₈ X ₉ GX ₁₀ SX ₁₁ KPX ₁₂ X _I3 NCX _I4 X ₁ - _j X _lft X ₁₇ X _Ill (SEQ ID NO 32), wherein X, is glycine or deleted, X ₆ is any amino acid residue, X ₇ is an ammo acid residue with an aliphatic side chain, X ₈ is a polar amino acid residue, X ₉ is an ammo acid residue with an aromatic side chain, X, ₀ is a polar ammo acid residue, X _M is any ammo acid residue, X ₎₂ is a polar amino acid residue or deleted, X _π is a basic amino acid residue or deleted, X _l4 is a small chain amino acid residue, X„ is any ammo acid residue, X, _ft is a basic or polar neutral amino acid residue, X ₁₇ is any amino acid residue, and X, ₈ is an aliphatic ammo acid residue More specifically, X ₆ is isoleucine, leucine, threonine, or valine, X ₇ is alanine, valine, isoleucine, or leucine, X ₈ is lysine, argmine, glutamine, or asparagine, X _q is phenylalanine, tryptophan, or tyrosine, X _l0 is threonine, serine, aspartic acid, or glutamic acid, X _π is leucine, isoleucine, methionine, or threonine, X, ₂ is serine or deleted, X _n is lysine, arginine, or deleted, X, ₄ is glycine or alanine, X _1S is asparagine, glutamine, glycine, serine, arginine, or lysine, X, is lysine, argmine, asparagine, or glutamine, X _π is lysine, arg ine, isoleucine, leucine, or valine. or X, _κ is alanine, valine, isoleucine, or leucine, or any combination of the foregoing

The present invention also advantageously provides for combining two overlapping peptides into a larger peptide having up to 35 amino acid residue In one such embodiment, the peptide has the ammo acid sequence

X _l BYCKIX ₂ CX,X ₄ GX,X ₆ HTX ₇ CX ₈ X„GX ₁₀ SX _{l l} KPX _l2 X _n NCX _l4 X _l ,X ₁₆ XπXι _S (SEQ ID NO 33), wherein X, is a neutral amino acid residue, X, is a basic am o acid residue or

deleted, X ₃ is any amino acid residue, X ₄ is a polar amino acid, X, is glycine or deleted, X ₆ is any amino acid residue, X ₇ is an ammo acid residue with an aliphatic side chain, X ₈ is a polar amino acid residue, X ₉ is an amino acid residue with an aromatic side chain, X, ₀ is a polar ammo acid residue, X _π is any amino acid residue, X _I2 is a polar ammo acid residue or deleted, X, ₃ is a basic ammo acid residue or deleted, X _l4 is a small chain amino acid residue, X„ is any amino acid residue, X _l6 is a basic or polar neutral amino acid residue, X, ₇ is any amino acid residue, and X _i8 is an aliphatic ammo acid residue In a more specific embodiment, X, is valine or asparagine, X ₂ is lysine, arginine, or deleted, X ₃ is pro ne, arginine, serine, or leucine, X ₄ is argmine, lysine, or serine, X ₆ isoleucine, leucine, threonine, or valme, X ₇ is alanine, valine, isoleucine, or leucine, X ₈ is lysine, arginine, glutamine, or asparagine, X ₉ is phenylalanine, tryptophan, or tyrosine, X, ₀ is threonine, serine, aspartic acid, or glutamic acid, X _n is leucine, isoleucine, methionine, or threonine, X _I2 is serine or deleted, X, ₃ is lysine, arginine, or deleted, X, ₄ is glycine or alanine, X,, is asparagine, glutamine, glycine, serine. arginine, or lysine, X _l is lysine, arginine, asparagine, or glutamine, X _π is lysine, argmine, isoleucine, leucine, or valine, or X, ₈ is alanine, val e, isoleucine, or leucine, or any combination of the foregoing

In another specific embodiment, the peptide has the amino acid sequence KX^XjoIX .X^X^HNX^FRQKX^AX^GLE (SEQ ID NO 34), wherein X ₁₉ is a basic or neutral polar am o acid residue X ₂₀ is any amino acid residue X ₂ , is an aliphatic amino acid residue. X ₂₂ is a polar amino acid residue, X ₂ , is a polar charged ammo acid residue, X ₂₄ is a polar amino acid residue, X _2< is an aliphatic amino acid residue, and X ₂₍ , is a polar basic or neutral amino acid residue More specifically, X, ₉ is asparagine, glutamine, or lysine, X ₂₀ is aspartic acid, glutamic acids, leucine, or isoleucine, X _2I is valme, leucine, or isoleucine, X ₂₂ arginine, asparagine, or serine, X ₂₃ is glutamic acid or arginine, X ₂₄ is aspartic acid, glutamic acid, asparagine, glutamine, or arginine, X ₂ , is valine, leucine, or isoleucine, or X ₂₆ is arginine, asparagine, or lysine. or any combination of the foregoing

In still another specific embodiment, the peptide has the amino acid sequence FRQKX ₂₅ AX ₂₆ GLETRGX ₂₇ PGPQPX ₂₈ (SEQ ID NO 35), wherein X _2S is an aliphatic ammo acid residue. X ₂₆ is a polar basic or neutral amino acid residue, X ₂₇ is a polar basic or neutral ammo acid residue, and X ₂₈ is any amino acid residue More specifically, X^ is valine, leucine, or isoleucine, X _2(l is argmine, asparagine, or lysine, X ₂₇ is asparagine or lysine, or X-, ₈ is glycine, alanine or pro ne, or anv combination of the foregoing

As noted above, overlapping peptides may be provided as a single, larger peptide Thus, in another embodiment, the peptide has the amino acid sequence

KX ₁₉ X ₂₀ IX ₂₁ X ₂₂ X ₂ HNX ₂₄ FRQKX ₂ ,AX ₂₆ GLETRGX ₂₇ PGPQPX ₂₈ (SEQ ID NO 36), wherein X, ₉ is a basic or neutral polar ammo acid residue X ₂₀ is any amino acid residue X _2I is an aliphatic amino acid residue, X ₂ is a polar amino acid residue, X ₂₃ is a polar charged amino acid residue, X ₂₄ is a polar ammo acid residue, X ₂ , is an aliphatic amino acid residue, X ₂₆ is a polar basic or neutral amino acid residue, X ₂₇ is a polar basic or neutral amino acid residue, and X ₂₈ is any amino acid residue More specifically. X ₁₉ is asparagine, glutamine, or lysine, X ₂₀ is aspartic acid, glutamic acids, leucine, or isoleucine, X ₂ , is valme, leucine, or isoleucine, X ₂₂ argmme, asparagine, or senne, X ₂₃ is glutamic acid or argmine, X ₂₄ is aspartic acid, glutamic acid, asparagine, glutamine, or argm e, X ₂₅ is valine, leucine, or isoleucine, X ₂₆ is argmme, asparagine, or lysine, X-, ₇ is asparagine or lysine, or X ₂₈ is glycine, alanine, or prohne, or any combination of the foregoing

In yet another embodiment, the peptide has the ammo acid sequence DELAX ₂₉ X ₃₀ AQX _3I WAX ₃₂ QCX ₃₃ X ₃₄ X ₃₅ X ₃₆ HD (SEQ ID NO 37), wherein X ₂₉ is any amino acid residue, X _3U is an aliphatic amino acid residue, X ₃ , is any amino acid residue, X ₃ is a polar neutral amino acid residue, X ₃₃ is a polar neutral or acidic amino acid residue, X ₃₄ is an aromatic amino acid residue, X _3< is an aliphatic am o acid residue or deleted, and X ₃₆ is any ammo acid residue More specifically, X ₂₉ is tyrosine, lysine, or histidine, X ₃₀ is isoleucine, leucine, or valme, X ₃ , is threonine or valine, X ₃₂ is serine or asparagine, X ₃₃ is glutamine, asparagine, aspartic acid, or serine, X ₃₄ is phenylalanine to tyrosine, X ₃ , is isoleucine, leucine, or deleted, or X _3f , is glycine, asparagine, or valme. or any combination of the foregoing

In still another specific embodiment, the peptide has the amino acid sequence NX ₃₇ X ₃₈ X ₃₉ X _4(J X ₄ ,X ₄₂ X ₄₃ X ₄₄ BX ₄ ,FX ₄₍ ,KX ₄₇ GHYTOM (SEQ ID NO 38), wherein X ₃₇ is a cyclic amino acid residue, X ₃₈ is a polar basic or neutral amino acid residue, X ₃₉ is any amino acid residue, X ₄₀ is any amino acid residue, X _4i is a non-polar ammo acid residue, X ₄₂ is any amino acid residue, X ₄ , is a polar ammo acid residue or glycine, X ₄₄ is a polar basic or neutral amino acid residue, X ₄ , is a polar neutral amino acid or deleted, X ₄₆ is any ammo acid residue, and X ₄₇ is a non-polar amino acid residue More specifically. X, ₇ is prohne or tyrosine, X ₃₈ is asparagine, arginine, or histidine, X _w is lysine, threonine. or

valine, X ₄₀ is aspartic acid, glycine, or lysine, X ₄ , is isoleucine, leucine, phenylalanine, or threonine, X ₄₂ is glutamine, isoleucine, leucine, methionine, serine, or threonine, X ₄₃ is asparagine, glutamic acid, histidine, lysine, or glycine, X^ is aspartic acid, asparagine, glutamine, or serine, X ₄₅ is asparagine or deleted, X _4ft is alanine, arginine, leucine, isoleucine, or serine, or X ₄₇ is isoleucine, leucine, threonine, or valine, or any combination of the foregoing

In still another embodiment, the peptide has the ammo acid sequence FX ₄₆ KX ₄₇ GHYTOMVWX ₄₈ X ₄₉ T (SEQ ID NO 39), wherein X ₄₆ is any amino acid residue, X ₄₇ is a non-polar amino acid residue, X ₄₈ is a small side chain amino acid residue, and X ₄₉ is a polar basic or neutral amino acid residue More specifically, X ₄ is alanine, arginine, leucine, isoleucine, or serine, X ₄₇ is isoleucine, leucine, threonine, or valine, X ₄₈ is glycine or alanine, or X ₄₉ is lysine or asparagine, or any combination of the foregoing

In another embodiment of the invention wherein two overlapping peptides are combined, the peptide has the amino acid sequence

NX, ₇ X ₃₈ X ₃₉ X ₄₀ X _4l X ₄₂ X ₄₃ X ₄₄ BX ₄₅ FX _4ή KX ₄₇ GHYTOMVWX ₄₈ X ₄₉ T (SEQ ID NO 40), wherein X ₃₇ is a cyclic amino acid residue, X ₃₈ is a polar basic or neutral amino acid residue, X ₃₉ is any am o acid residue, X ₄₀ is any amino acid residue, X ₄ , is a non-polar amino acid residue, X ₄₂ is any am o acid residue, X ₄₃ is a polar amino acid residue or glycine, X^ is a polar basic or neutral amino acid residue, X _4< is a polar neutral ammo acid or deleted, X^ is any amino acid residue, X ₄₇ is a non-polar amino acid residue, X ₄₈ is a small side chain amino acid residue, and X ₄₉ is a polar basic or neutral amino acid residue More specifically, X ₃₇ is prohne or tyrosine, X ₃₈ is asparagine, arginine, or histidine, X ₃₉ is lysine, threonine, or valine, X^ is aspartic acid, glycine. or lysine, X ₄ , is isoleucine, leucine, phenylalanine, or threonine, X ₄₂ is glutamine, isoleucine, leucine, methionine, serine, or threonine, X ₄ , is asparagine, glutamic acid, histidine. lysine, or glycine, X^ is aspartic acid, asparagine, glutamine, or serine, X _4S is asparagine or deleted X ₄₆ is alanine. argmme, leucine, isoleucine, or serine, X ₄₇ is isoleucine, leucine, threonine, or valine, X ₄₈ is glyc e or alanme, or X ₄₉ is lysine or asparagine, or any combination of the foregoing

In still another embodiment, the peptide has the amino acid sequence X _w ZX ,X, ₂ X ₅₃ X, ₄ X ₅ ,HYLX _5f ,CNYGPX ₅₇ GNX ₅₈ X, ₉ X _N) (SEQ ID NO 40), wherein X _5π is a

non-polar amino acid, X _5I is a polar acidic or neutral amino acid residue, X ₅₂ is a polar basic or neutral ammo acid residue, X ₅₃ is a non-polar amino acid residue, X, ₄ is a moderately polar ammo acid residue, X ₅ , is a polar basic or neutral ammo acid residue, X ₅₆ is an aliphatic amino acid residue, X ₅₇ is any amino acid residue, X, is any ammo acid residue, X, ₉ is any amino acid residue, and X _(l0 is any amino acid residue More specifically, X ₅₀ is isoleucine, leucine, or methionine, X ₅₁ is asparagine, aspartic acid, glutamine, or glutamic acid, X ₅₂ is asparagine or lysine, X ₅₃ is methionine or tryptophan, X ₅₄ glutamine, histidine, or tyrosine, X ₅₅ is asparagine, lysme, or threonine, X, ₆ isoleucine, leucine, or valine, X, ₇ is alanine, glycine, or serine, X ₅₈ is aspartic acid, phenylalanine, or tyrosine, X ₅₉ is glutamine, leucine, isoleucine, methionine, or phenylalanine, or X _w is asparagine, aspartic acid, or glycine, or any combination of the foregoing

In specific embodiments, the invention is directed to a peptide having an ammo acid sequence selected from the group consisting of

NNYCKIKCRKGIHTLCKFGT (SEQ ID NO 8), or its homologue, GIHTLCKFGTSMKPNCGRNV (SEQ ID NO 9), or its homologue, NNYCKIKCRKGIHTLCKFGTGTSMKPNCGRNV (SEQ ID NO 42), or its homologue, KNEILKRHNDFRQNVAKGLE (SEQ ID NO 12), or its homologue,

FRQNVAKGLETRGKPGPQPP (SEQ ID NO 13), or its homologue, KNEILKRHNDFRQNVAKGLETRGKPGPQPP (SEQ ID NO 43), or its homologue, DELAKIAQTWANQCDFNHDD (SEQ ID NO 16), or its homologue, NYKVGLQNSNFRKVGHYTQM (SEQ ID NO 22), or its homologue,

FRKVGHYTQMVWGKT (SEQ ID NO 23), or its homologue, NYKVGLQNSNFRKVGHYTQMVWGKT (SEQ ID NO 44), or its homologue, and IEDNWYTHYLVCNYGPGGND (SEQ ID NO 26), or its homologue,

In specific embodiments, infra, peptides having SEQ ID NOS 8, 9, 12, 13, 16, 22, 23, and 26 are shown to be dominant in more than one strain of mouse

In addition to homologous variants, allehc variant, consensus variant, and combined variant peptides of the invention, the present invention further provides a recombinant polypeptide comprising two or more peptides non-contiguously arranged relative to the native sequence of vespid venom antigen 5 Although the term recombinant generally refers to polypeptides generated by genetic engineering, which meaning is fully intended according to the present invention, the term recombinant is used herein more generally, to refer to polypeptides created by combination (or recombination) of non-contiguous immunodominant peptide fragment of vespid venom antigen 5 Such fragments may be from antigen 5 from the same species, antigen 5 peptides from different species, consensus antigen 5 peptides as described above, or any combination of the foregoing In a specific embodiment, the peptides are selected from the group consisting of

NNYCKIKCRKGIHTLCKFGT (SEQ ID NO 8), or its homologue, GIHTLCKFGTSMKPNCGRNV (SEQ ID NO 9), or its homologue, NNYCKIKCRKGIHTLCKFGTGTSMKPNCGRNV (SEQ ID NO 42), or its homologue,

KNEILKRHNDFRQNVAKGLE (SEQ ID NO 12), or its homologue, FRQNVAKGLETRGKPGPQPP (SEQ ID NO 13), or its homologue, KNEILKRHNDFRQNVAKGLETRGKPGPQPP (SEQ ID NO 43), or its homologue, DELAKIAQTWANQCDFNHDD (SEQ ID NO 16), or its homologue,

NYKVGLQNSNFRKVGHYTQM (SEQ ID NO 22), or its homologue, FRKVGHYTQMVWGKT (SEQ ID N0.23), or its homologue, NYKVGLQNSNFRKVGHYTQMVWGKT (SEQ ID NO 44), or its homologue, and IEDNWYTHYLVCNYGPGGND (SEQ ID NO 26), or its homologue

In specific embodiments, the present invention is directed to the following homologous T cell peptides of hornet Ag5 from two species each of hornet (Doi a and Doi m), yellowjacket (Ves m and Ves v) and wasp (Pol a and Pol e) and one species of fire ant (Sol i) shown in Table 2 As noted above, there are two forms of Doi m 5 01 and 5 02, referred to as Doi m A and B here Residues given below refer to Doi m 5.02 numbering, and gaps are added to obtain maximal sequence alignment Where immunodominant peptides overlap, the fragment containing the overlapping sequences are provided, with the specific sequences demarcated by a line under the peptide group In

such specific embodiments, the present invention contemplates both the individual immunodominant peptides and the larger peptide containing the overlapping segments.

Table 2. Homologues of Immunodominant Peptides

Peptides 1 and 2 (residue 1-20 and 11-30)

Ves m NNYCKI KCLKGG VHTACKYG SLKP NCGNKkV SEQ ID NO:45 Ves v NNYCKI KCLKGG VHTACKYG SLKP NCGNKvV SEQ ID NO: 46 Doi a NNYCKI CpKG tHTLCKYGTSMKP NCGgKIVKSEQ ID NO:47 Doi mA NNYCKI KCsrG IHTLCKFGTSMKP NCGsKIVKSEQ ID NO:48 Doi TΠB NNYCKI KCrkG IHTLCKFGTSMKP NCGrnVVKSEQ ID NO: 49 Pol a VDYCKI KCPSG IHTVCQYGESTKPSKNCAGKVIKSEQ ID NO: 50 Pol e VDYCKI KCPSG IHTVCQYGESTKPSKNCAGKVIKSEQ ID NO: 51 Sol i YNYCNLQSCKRNNAIHTMCQY TSPTPGPMCLEYSN SEQ ID NO: 52

2a

5 and 6 (residue 41-60 and 51-70)

KQDILKEHNDFRQKIARGLETRGNPGPQPPA KQDILKEHNDFRQKIARGLETRGNPGPQPPA KNEIVKRHNEFRQKVAqGLETRGNPGPQPPA KNEIVnRHNQFRQKVAKGLETRGNPGPQPPA KNEIlkRHNDFRQnVAKGLETRGkPGPQPPA KKLIVSEHNRFRQKVAQGLETRGNPGPQPAA KKLIVSEHNRFRQKVAQGLETRGNPGPQPAA KDAIVNKHNELRQRVASGKEMRGTNGPQPPA

Peptide 9 {residue 81-100)

DELAYiAQVWANQCQY GHDT DELAYvAQV ANQCQY GHDT DELAKIAQT ANQCnF GHDQ DELAKIAQTWANQCsF GHDQ DELAKIAQTWANQCdF nHDD DELAHIAQVWASQCQFL VHDK DELAHIAQVWASQCQFL VHDK PELATIAQRWANQCTE EHDA

idue 141-160 and 151-165)

FLKiGHYTQMVWANT SEQ ID NO: 68

FLKtGHYTQMV ANT SEQ ID NO: 69

FSKVGHYTQMVWGKT SEQ ID NO:70

FSKVGHYTQMVWGKT SEQ ID NO: 71

FrKVGHYTQMVWGKT SEQ ID NO: 44 FAKIGHYTQMV GKT SEQ ID NO: 72 FAKIGHYTQMV GKT SEQ ID NO: 73

Sol i NYNTGISFPSDDNILMKVEHYTQIV AKT SEQ ID NO: 74

15

20

Peptide 18 (residue 176-1951

Ves m IQEn HKHYLVCNYGPSGNF SEQ ID NO: 75

Ves v IQEK HKHYLVCNYGPSGNF SEQ ID NO: 76

Doi a IENKWHTHYLVCNYGPAGNY SEQ ID NO: 77

Doi mA IENNWHTHYLVCNYGPAGNY SEQ ID NO: 78 Doi mB IEdNWyTHYLVCNYGPgGNd SEQ ID NO:26

Pol a mENNMQNHYLICNYGPAGNY SEQ ID NO: 79

Pol e iENkMQNHYLICNYGPAGNY SEQ ID NO: 80

Sol i EPDN TKHYLVCNYGPAGNV SEQ ID NO: 81

The present invention further provides a pharmaceutical composition for treating vespid venom sensitivity, preferably where sensitivity to antigen 5 has been demonstrated, comprising any of the foregoing peptides of the invention and a pharmaceutically acceptable carrier. In a preferred aspect, the pharmaceutical composition comprises more than one peptide of the invention, thus greatly increasing the breadth of its effectiveness.

The invention naturally extends as well to a method for treating sensitivity to vespid venom comprising administering to a vespid venom allergic patient a therapeutically effective amount of a pharmaceutical composition of the invention. Preferably, the patient has been identified as sensitive to vespid venom antigen 5.

As demonstrated herein, the peptides of the invention are cross-reactive for a native testes protein. Thus, an important advance of the present invention is that it provides peptides specific for treatment of antigen 5-sensitive patients, regardless of the cause of this antigen 5 sensitivity. In other words, as demonstrated herein, whether a subject develops antigen 5 sensitivity from vespid stings, fire ant bites, or to testes protein, the peptides of the present invention provide an advantageous therapeutic agent for treating such sensitivity.

Thus, a primary object of the present invention is to provide immunodominant peptides from vespid venom antigen 5.

A corollary object is to provide such immunodominant peptides that produce T cell anergy in subjects sensitive or allergic to vespid venom antigen 5.

Yet another object of the invention is to provide combinations of such immunodominant peptides for a broad treatment of vespid venom sensitive patients from a wide variety of MHC (HLA) backgrounds.

Still another object of the invention is to provide consensus immunodominant T cell epitopes of vespid venom antigen 5s.

These and other objects of the present invention can be better appreciated by reference to the following drawings, the Detailed Description of the Invention, and the Examples.

ABBREVIATIONS

Ag5, Ag 5 antigen 5

Doi m Dolichovespula maculata white face hornet Doi a D. arenaria yellow hornet

Pol a Polistes annularis wasp

Pol e P. exclamans wasp

Ves tn Vespula maculifrons yellowjacket

Ves v V. vulgaris yellowjacket n-, r- natural, recombinant (respectively)

PCR polymerase chain reaction

TCR T cell receptor for antigen tpx testes protein

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. Amino acid sequence of vespid antigen 5s. Antigen 5s were isolated from yellowjackets (Vespula maculifrons and vulgaris), hornets (Dolichovespula arenaria and maculata) and wasps (Polistes annularis and exclamans). The sequences given are in the

order of Ves m V, Ves v V, Doi a V, Doi m VA and VB, Pol a V, and Pol e V respectively Residues which are common to all sequences are given on separate lines beneath the sequences, residues underlined or dotted represent conserved or variable regions, respectively

FIGURE 2 Peptide fragments of Doi m Antigen 5 2

FIGURE 3 Sequence similarity of hornet Ag5 and mouse and human tpx ^' s Bold characters indicate those residues of human and mouse tpx's which are identical with those of Ag5 The underlines regions of greater than 7 residues in length have greater than 67% sequence identity, and each is interspersed with fewer than 4 substituted residues

FIGURE 4 Stimulation index profile of BALB/c mice spleens after 4 immunizations with natural (n)-Ag5 In vitro proliferation assays were performed with peptides at three concentrations 1 x 10 ⁵ M (open bar), 1 25 x 10 ⁶ M ("uphill" hatch), and 1 56 x 10 ⁷ M ("downhill" hatch) The corresponding concentrations of recombinant antigen 5 control were 1 0 x 10 ⁶ M, 1 25 x 10 ⁷ , and 1 56 x 10 ⁸ M The blank was 2200 cpm

FIGURE 5 Stimulation index profile of BALB/c mice spleens after 4 immunizations with recombinant (r)-Ag5 In vitro proliferation assays were performed with peptides at three concentrations 1 x 10 ^ M (open bar), 1 25 x 10 M (' uphill" hatch) and 1 56 x 10 ⁷ M ("downhill" hatch) The corresponding concentrations of recombinant Antigen 5 were 1 0 x 10 ⁶ M. 1 25 x 10 ⁷ , and 1 56 x 10 * M The blank was 4700 cpm

FIGURE 6 Stimulation index profile of BALB/c mice spleens after 4 immunizations with r-Ag5 In vitro proliferation assays were performed with peptides at three concentrations 1 x 10 ⁵ M (open bar), 1 25 x 10 ⁶ M ("uphill" hatch), and 1 56 x 10 ⁷ M (" , wnhill" hatch) The corresponding concentrations of recombinant Antigen 5 were 1 0 x 10 ⁶ M, 1 25 x 10 ⁷ , and 1 56 x 10 ⁸ M The blank was 3970 cpm

FIGURE 7 Stimulation index profile of BALB/c mice spleens after 4 immunizations with r-frag IN (residue 1-114 of antigen 5) In vitro proliferation assays were performed with peptides at three concentrations 1 x 10 ⁵ M (open bar), 1 25 x 10 ^b M (' uphill"

hatch), and 1.56 x 10 ^"7 M ("downhill" hatch). The corresponding concentrations of recombinant Antigen 5 were 1.0 x 10" M, 1.25 x 10 ⁷ , and 1.56 x 10 ⁸ M. The blank was 4690 cpm.

FIGURE 8. Stimulation index profile of BALB/c mice spleens after 4 immunizations with r-frag C (residue 151-204 of antigen 5). In vitro proliferation assays were performed with peptides at three concentrations: 1 x 10 ^'5 M (open bar), 1.25 x 10" M ("uphill" hatch), and 1.56 x 10 ^'7 M ("downhill" hatch). The corresponding concentrations of recombinant Antigen 5 were 1.0 x 10" M, 1.25 x 10 ⁷ , and 1.56 x 10 ⁸ M. The blank was 6620 cpm.

FIGURE 9. Stimulation index profile of BALB/c mice spleens after 5 immunizations with r-frag IN. In vitro proliferation assays were performed with peptides at three concentrations: 1 x 10 ^s M (open bar), 1.25 x 10" M ("uphill" hatch), and 1.56 x 10 ^"7 M ("downhill" hatch). The corresponding concentrations of recombinant Antigen 5 were 1.0 x 10" M, 1.25 x 10 ⁷ , and 1.56 x 10 ^"8 M. The blank was 4410 cpm.

FIGURE 10. Stimulation index profile of BALB/c mice spleens after 5 immunizations with r-frag C. In vitro proliferation assays were performed with peptides at three concentrations: 1 x 10 ⁵ M (open bar), 1.25 x 10" M ("uphill" hatch), and 1.56 x 10' ⁷ M ("downhill" hatch). The corresponding concentrations of recombinant Antigen 5 were 1.0 x 10" M, 1.25 x 10 ⁷ , and 1.56 x 10 ⁸ M. The blank was 7520 cpm.

FIGURE 11. Stimulation index profile of AS W/sn mice spleens after 5 immunizations with r-Ag5. In vitro proliferation assays were performed with peptides at three concentrations: 1 x 10 ⁵ M (open bar), 1.25 x 10" M ("uphill" hatch), and 1.56 x 10 ⁷ M ("downhill" hatch). The corresponding concentrations of recombinant Antigen 5 were 1.0 x 10" M, 1.25 x 10 ⁷ , and 1.56 x 10 ⁸ M. The blank was 5640 cpm.

FIGURE 12. Stimulation index profile of P/J mice spleens after 5 immunizations with r-Ag5. In vitro proliferation assays were performed with peptides at three concentrations: 1 x 10 ^"5 M (open bar), 1.25 x 10" M ("uphill" hatch), and 1.56 x 10 ^"7 M ("downhill" hatch). The corresponding concentrations of recombinant Antigen 5 were 1 .0 x 10" M, 1.25 x 10 ^'7 , and 1.56 x 10 ^"8 M. The blank was 1730 cpm.

FIGURE 13 Stimulation index profile of C3H/He mice spleens after 5 immunizations with r-Ag5 In vitro proliferation assays were performed with peptides at three concentrations 1 x 10 ⁵ M (open bar), 1 25 x 10 ⁶ M ("uphill" hatch), and 1 56 x 10 ⁷ M ("downhill" hatch) The corresponding concentrations of recombinant Antigen 5 were 1 O x 10 ⁶ M, 1 25 x 10 ⁷ , and 1 56 x 10 ⁸ M The blank was 3130 cpm

FIGURE 14 Stimulation index profile of C57B1/6 mice spleens after 5 immunizations with r-Ag5 In vitro proliferation assays were performed with peptides at three concentrations. 1 x 10 ⁵ M (open bar), 1 25 x 10 " M ("uphill" hatch), and 1 56 x 10 ⁷ M ("downhill" hatch) The corresponding concentrations of recombinant Antigen 5 were 1 0 x 10 ⁶ M, 1 25 x 10 ⁷ , and 1 56 x 10 ⁸ M The blank was 1050 cpm

FIGURE 15 Stimulation index profile of BALB/c mice spleen cells after 4 immunizations with Tpx-N Stimulation was tested with peptide or protein at three different concentrations Open bars - 1 0 x 10 ⁵ M peptide or Frag IN, 2 5 x 10 ⁶ M Tp X-N, "uphill " hatch - 1 25 x 10 " M peptide or Frag IN, 3 13 x 10 ⁷ M, Tpx-N, "downhill" hatch - 1 5 x 10 ⁷ M peptide and Frag IN, 3 91 x 10 M Tpx-N, and stippled -- 1 96 x 10 ⁸ M Frag IN, 4 89 x 10 " M Tpx-N The blank was 7270 cpm

FIGURE 16 Stimulation index profile of BALB/c mice spleen cells after 4 immunizations with Tpx-C Stimulation was tested with peptide or protein at three different concentrations Open bars - 1 x 10 M peptide, 5 x 10 ⁶ M Frag C or Tpx-C, "uphill" hatch - 1 25 x 10 ⁶ M peptide, 6 25 x 10 ⁷ M Frag C or Tpx - C, "downhill ' hatch - 1.56 x 10 ⁷ M peptide, 7 8 x 10 ⁸ M Frag C, Tpx - C, and stippled -- 9 75 x 10 " M Frag - C or Tpx - C The blank was 5110 cpm

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides the sequences of immunodominant peptides of vespid venom antigen 5, and corresponding peptides from other antigens, such as fire ant Sol l 3

The term "immunodominant peptide" is used herein to refer to a peptide that contains a T cell epitope of the antigen, such that T cells immunized with the antigen will be stimulated

when contacted with the peptide The term T cell epitope is used herein in conformity with the definition in immunology In one embodiment, an immunodominant epitope is an epitope that induces a greater than about 4-fold increase in stimulation of immunized T cells in an in vitro stimulation assay, particularly a greater than 2-fold, preferably greater than 4-fold, and more preferably greater than 6-fold increase in the stimulation index m a Η-thymidine incorporation assay In another embodiment, an immunodominant peptide is a peptide that induces proliferation or stimulation of different MHC-restπcted populations of immunized T cells

An immunodominant peptide of the invention may contain one epitope, or it may contain more than one epitope

An lmmunomodulatory peptide" is a peptide that affects immune response in vivo Preferably, an lmmunomodulatory peptide of the invention induces T cell anergy when administered to a subject

The invention also provides pharmaceutical compositions effective for the treatment of a vespid venom allergen-specific allergic condition comprising a polypeptide of the invention, and methods for treating such allergic conditions comprising administering a therapeutical ly effective dose of the pharmaceutical compositions of the invention

The polypeptides of the invention can also be useful for diagnosis of vespid venom- specific allergic conditions

As used herein, the term "vespid venom allergen" refers to a protein found in the venom of a vespid, to which susceptible people are sensitized on exposure to the sting of the insect While most antigens are characterized by being reactive with specific IgG class antibodies, an allergen is characterized by also being reactive with IgE type antibodies The IgE type antibodies are responsible for mediating the symptoms of an allergic condition, i e . immediate-type hypersensitivity According to the invention, the vespid venom allergen of interest is vespid venom antigen 5

As herein, the term "vespid" is used according to the practice of those m 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 Po stinae 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 vulgans (L ), and V pennsylvamca (Saussure) Species in the genus Polistes include but are not limited to P annularis (Linnaeus), P exclamans (Viereck) P metncus (Say), P fuscatus (Fabπcius), 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 orienta s (Linnaeus)

As used herein, the term "lmmunomodulatory" refers to an ability to increase or decrease an antigen-specific immune response, either at the B cell or T cell level lmmunomodulatory 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 T cell responses, the lmmunomodulatory polypeptides of the invention may bind to immunoglobulin (i e . antibody) molecules on the surface of B cells, and affect B cell responses as well

For the sake of clarity, the present invention is described in detail in sections relating to an lmmunomodulatory fragment of a vespid venom antigen 5, or derivatives and analogs of the vespid venom antigen 5, assays with the lmmunomodulatory vespid venom antigen 5, fragments, or derivatives or analogs thereof, and finally therapeutic and diagnostic uses of the vespid venom antigen 5 fragments, or derivatives or analogs thereof

Immunodominant Peptides of Vespid Venom Ag5 An immunodominant peptide of the invention is a peptide comprising at least one T cell epitope, with a minimum size of approximately 8 amino acid residues (the minimum length generally regarded as requisite for antigen-specific recognition by a T cell) A peptide comprising at least one T cell epitope is capable of eliciting a T cell response, such as stimulation or T cell anergy (toleπzation)

The immunodominant peptides of the invention have been identified muπne backgrounds However, those peptides that are antigenic (comprise a T cell epitope) in

more than one mouse strain are ^' excellent candidates for immunodominant epitopes in antigen 5 sensitive humans. As pointed out above, on the basis of findings with Fel d 1 and Der p 1 , it is reasonable to conclude that the T cell epitope data of a vespid venom antigen 5 found in mice, particularly in multiple strains, will be applicable for humans.

Also contemplated by the term "immunodominant peptide" are peptides which join two or more discontinuous epitopes (or the specific peptides disclosed herein) in a single polypeptide. In one aspect, such "aggregate" polypeptides can be prepared chemically, e.g. , by crosslinking peptides, or using condensation techniques to form peptide bonds. Alternatively, synthetic DNA molecules encoding such aggregate peptides can be prepared, inserted in a recombinant expression vector, and the aggregate peptide expressed by host cells transformed or transfected with the expression vector and cultured under conditions that allow for expression of the aggregate polypeptide.

Immunodominant peptides of the invention can also be prepared as fusion proteins.

Amino acids used for peptide synthesis may be standard Boc (N ^α -amino protected N"-t- butyloxycarbonyl) amino acid resin with the standard deprotecting, neutralization, coupling and wash protocols of the original solid phase procedure of Merrifield [1963, J. Am. Chem. Soc. 85:2149-2154], or the base-labile N"-amino protected

9-fluorenylmethoxycarbonyl (Fmoc) amino acids first described by Carpino and Han [1972, J. Org. Chem. 37:3403-3409]. Both Fmoc and Boc N ^π -amino protected amino acids can be obtained from Fluka, Bachem, Advanced Chemtech, Sigma. Cambridge Research Biochemical, Bachem, or Peninsula Labs or other chemical companies familiar to those who practice this art. In addition, the method of the invention can be used with other N ⁿ -protecting groups that are familiar to those skilled in this art. Solid phase peptide synthesis may be accomplished by techniques familiar to those in the art and provided, for example, in Stewart and Young, 1984, Solid Phase Synthesis, Second Edition, Pierce Chemical Co. , Rockford, IL; Fields and Noble. 1990, Int. J. Pept. Protein Res. 35: 161-214, or using automated synthesizers, such as sold by ABS.

Alternatively., immunodominant peptides of the invention can be prepared by recombinant DNA techniques. Thus, 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 II [D.N. Glover ed. 1985]; Oligonucleotide Synthesis [M.J. Gait ed. 1984]; Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal. A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994)].

Peptides comprising at least two regions, each region comprising at least one T cell epitope of a antigen 5 are also within the scope of the invention. Each region of such peptides is derived from the same or from different antigen 5. Isolated peptides. each comprising at least two T cell epitopes of antigen 5, are particularly desirable for increased therapeutic effectiveness. Peptides immunologically related by T cell cross- reactivity, which are capable of reacting with the same T cells as a peptide of the invention, are also contemplated.

As noted above, isolated peptides of the invention can be produced by recombinant DNA techniques in a host cell transformed with a nucleic acid having a sequence encoding such peptide. The isolated peptides of the invention can also be produced by chemical synthesis. In certain limited situations, isolated peptides can be produced by chemical cleavage of the protein allergen. When a peptide is produced by recombinant techniques, host cells transformed with a nucleic acid having a sequence encoding the peptide or the functional equivalent of the nucleic acid sequence are cultured in a medium suitable for the cells and peptides can be purified from cell culture medium, host cells, or both using techniques known in the art for purifying peptides and proteins including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, or immunopurification with antibodies specific for the peptide, antigen 5 from which the peptide is derived, or a portion thereof. Isolated peptides of the invention are substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when synthesized chemically.

Naturally, the present invention provides expression vectors and host cells transformed to express the peptides. A nucleic acid sequence coding for a peptide of the invention may be expressed in bacterial cells such as E. coli, insect cells (baculovirus), yeast, or mammalian cells such as Chinese hamster ovary cells (CHO). Suitable expression vectors, promoters, enhancers, and other expression control elements may be found in Sambrook et al. Molecular Cloning: A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York ( 1989). Other suitable expression vectors, promoters, enhancers, and other expression elements are known to those skilled in the art. Many yeast and bacterial vectors are commercially available or on deposit at various depositories. Baculovirus and mammalian expression systems are also available.

For expression in E. coli, suitable expression vectors include, among others, pTRC [Amann et al., Gene, 69:301-315, (1988)], pGEX (Amrad Corp., Melbourne, Australia); pMAL (N.E. Biolabs, Beverly, MA); pRIT5 (Pharmacia, Piscataway, NJ); pET-l ld (Novagen, Madison, WI) [Jameel et al. , J. Virol. 64:3963-3966 (1990)]; pQE12 (QIAGEN, Chatsworth, CA); and pSEM [Knapp et al., BioTechniques , 8:280-281 (1990)]. The use of pTRC, and pET-l ld, for example, will lead to the expression of unfused protein. The use of pMAL, pRIT5, pSEM, pQE12, and pGEX will lead to the expression of allergen fused to maltose E binding protein (pMAL), protein A (pRIT5), truncated β-galactosidase (PSEM), hexahistidine (pQE12), or glutathione S-transferase (pGEX). When a polypeptide allergen is expressed as a fusion protein, it is particularly advantageous to introduce an enzymatic cleavage site at the fusion junction between the carrier protein and a polypeptide. The immunodominant polypeptide (harboring the peptide of the invention) may then be recovered from the fusion protein through enzymatic cleavage at the enzymatic site and biochemical purification using conventional techniques for purification of proteins and peptides. Suitable enzymatic cleavage sites include those for blood clotting Factor Xa or thrombin for which the appropriate enzymes and protocols for eavage are commercially available from, for example, Sigma Chemical Company, St. Louis, MO and N.E. Biolabs, Beverly, MA. The different vectors also have different promoter regions allowing constitutive or inducible expression with, for example, IPTG induction (PRTC [Amann et al. , (1988), supra]; pET-l ld, Novagen, Madison, WI) or temperature induction (pRIT5, Pharmacia, Piscataway, NJ).

Human T cell stimulating activity can be tested by culturing T cells obtained form an individual sensitive to vespid venom (. e , an individual who has an IgE mediated immune response to antigen 5) with an immunodominant peptide of the invention and determining whether proliferation of T cells occurs in response to the peptide as measured, e g , by cellular uptake of tπtiated thymidine Stimulation indices for responses by T cells to peptides can be calculated as the maximum CPM in response to a peptide divided by the control CPM A T cell stimulation index (S I ) equal to or greater than two times the background level is considered ' positive ' Positive results are used to calculate the mean stimulation index for each peptide for the group of peptides tested Peptides of this invention comprise at least one T cell epitope and have a mean T cell stimulation index of greater than or equal to 2 0 Preferably the S I value is greater than four A peptide having a T cell stimulation index of greater than or equal to 2 0, and more preferably greater than or equal to 4 0, is considered useful as a therapeutic agent Preferred peptides have a mean T cell stimulation index of at least 2 5, more preferably at least 3 5, even more preferably at least 4 0, and most preferably at least 5 0

In addition, preferred peptides have a positivity index (P I ) of at least about 100, more preferably at least 150, even more preferably at least about 200 and most preferably at least about 250 The positivity index for a peptide is determined by multiplying the mean T cell stimulation index by the percent of individuals, in a population of individuals sensitive to vespid venom (e g , preferably at least 9 individuals, more preferably at least 16 individuals or more, more preferably at least 29 individuals or more, or even more preferably at least 30 individuals or more), who have T cells that respond to the peptide Thus, the positivity index represents both the strength of a T cell response to a peptide (S I ) and the frequency of a T cell response to a peptide in a population of individuals sensitive to vespid venom

In order to determine precise T cell epitopes by, for example, fine mapping techniques, a peptide having T cell stimulating activity and thus comprising at least one T cell epitope as determined by T cell biology techniques is modified by addition or deletion of amino acid residues at either the ammo or carboxyl terminus of the peptide and tested to determine a change in T cell reactivity to the modified peptide If two or more peptides which share an area of overlap in the native protein sequence are found to have T cell stimulating activity, preferably human T cell stimulating activity, as determined by T cell biology

techniques, additional peptides can be produced comprising all or a portion of such peptides and these additional peptides can be rested by a similar procedure. Following this technique, peptides are selected and produced recombinantly or synthetically. Peptides are selected based on various factors, including the strength of the T cell response to the peptide (e.g. , stimulation index), the frequency of the T cell response to the peptide in a population of individuals sensitive to vespid venom, and the potential cross-reactivity of the peptide with other antigen 5 homologues. The physical and chemical properties of these selected peptides (e.g. , solubility, stability) are examined to determine whether the peptides are suitable for use in therapeutic compositions or whether the peptides require modification as described herein. The ability of the selected peptides or selected modified peptides to stimulate human T cells (e.g. , induce proliferation, lymphokine secretion) is determined.

Additionally, preferred peptides of the invention do not bind immunoglobulin E (IgE), or bind IgE to a substantially lesser extent than the protein allergen from which the peptide is derived binds IgE. The major complications of standard immunotherapy are IgE-mediated response such as anaphylaxis. Immunoglobulin E is a mediator of anaphylactic reactions which result from the binding and cross-linking of antigen to IgE on mast cells or basophils and the release of mediators (e.g. , histamine, serotonin, eosinophil chemotactic factors). Thus, anaphylaxis in a substantial percentage of a population of individuals sensitive to vespid venom could be avoided by the use in immunotherapy of a peptide of peptides which do not bind IgE in a substantial percentage (e.g. , at least about 75 %) of a population of individuals sensitive to vespid venom allergen, or if the peptide binds IgE, such binding does not result in the release of mediators from mast cells or basophile. The risk of anaphylaxis could be reduced by the use in immunotherapy of a peptide or peptides which have reduced IgE binding. Thus, peptides which have minimal IgE stimulating activity are desirable for therapeutic effectiveness. Minimal IgE stimulating activity refers to IgE production that is less than the amount of IgE production and/or IL-4 production stimulated by the native protein allergen.

A peptide of the invention, when administered to a vespid venom-sensitive individual, is capable of modifying the allergic response of the individual to the allergen. Particularly, peptides of the invention comprising at least one T cell epitope of antigen 5 when administered to an individual sensitive to vespid venom are capable of modifying T cell

response of the individual to the allergen As used herein, modification of the allergic response of an individual to vespid venom antigen 5 can be defined as non-responsiveness or diminution in symptoms upon exposure to antigen 5, as determined by standaid clinical procedures [see e g , Varney et al , British Medical Journal, 302 265-169 (1990)], including diminution in antigen 5 induced asthmatic symptoms As referred to herein, a diminution in symptoms includes any reduction in allergic response of an individual to the allergen after the individual has completed a treatment regimen with a peptide or protein of the invention This diminution may be subjective (i e , the patient feels more comfortable in the presence of the allergen) Diminution in symptoms can be determined clinically as well, using standard skin tests as is known in the art

As a result of the work described herein, peptides derived from antigen 5 comprising at least one T cell epitope can be produced T cell epitopes are believed to be involved in initiation and perpetuation of the immune responses to allergen(s), which are responsible for the clinical symptoms of vespid venom These T cell epitopes are thought to trigger early events at the level of the T helper cell by binding to an appropriate HLA (MHC) molecule on the surface of an antigen presenting cell and stimulating the relevant T cell subpopulation These events lead to T cell proliferation, lymphokine secretion, local inflammatory reactions, the recruitment of additional immune cells to the site, and activation of the B cell cascade leading to production of antibodies One isotype of these antibodies, IgE, is fundamentally important m the development of allergic symptoms and its production is influenced early in the cascade of events, at the level of the T helper cell, by the nature of the lymphokines secreted A T cell epitope is the basic element or smallest unit of recognition by a T cell receptor where the epitope comprises amino acid residues essential to receptor recognition Amino acid sequences which mimic those of T cell epitopes and which modify the allergic response to vespid venom antigen 5 are within the scope of this invention

Exposure of vespid venom allergic patients to peptides of the present invention may toleπze or anergize appropriate T cell subpopulations such that they become unresponsive to vespid venom allergen(s), e g , antigen 5, and do not participate m mounting an immune response upon such exposure In addition, administration of a peptide of the present invention may modify the lymphokine secretion profile as compared with exposure to the naturally-occurring vespid venom allergm or portion thereof (e g , result in a

decrease of IL-4 and/or an increase in IL-2). Furthermore, exposure to a peptide of the invention may influence T cell subpopulations which normally participate in a response to vespid venom allergen(s) such that these T cells are drawn away from the site(s) of normal exposure to the allergen (e.g. , nasal mucosa, skin, and lung) towards the site(s) of therapeutic administration of the peptide. This redistribution of T cell subpopulations may ameliorate or reduce the ability of an individual's immune system to mount an immune response at the site of normal exposure to the antigen 4, resulting in a diminution in allergic symptoms.

Isolated peptides of the invention comprise at least one T cell epitope of vespid venom antigen 5 and accordingly, the peptide comprises at least approximately eight amino acid residues of the protein allergen. For purposes of therapeutic effectiveness, therapeutic compositions of the invention preferably comprise at least two T cell epitopes of antigen 5. Accordingly, isolated peptides of the invention preferably comprise at least two T cell epitopes and accordingly, the peptide comprises at least approximately nine amino acid residues, and preferably at least 15 amino acid residues. Additionally, therapeutic compositions of the invention preferably comprise a sufficient percentage of the T cell epitopes of the entire protein allergen such that a therapeutic regimen of administration of the composition to an individual sensitive to vespid venom results in T cells of the individual being tolerized to the protein allergen. Synthetically produced peptides of the invention comprising up to approximately 35 amino acid residues in length, and more preferably up to approximately 20 amino acid residues in length are particularly desirable. Increases in length beyond this point may result in difficulty in peptide synthesis as well as retention of an undesirable property (e.g. , immunoglobulin binding or enzymatic activity) due to maintenance of conformational similarity between the peptide and the protein allergen from which it is derived.

Another embodiment of the present invention provides peptides comprising at least two regions, each region comprising at least one T cell epitope of vespid venom antigen 5 and accordingly, each region comprises at least approximately seven amino acid residues.

These peptides comprising at least two regions can comprise as many amino acid residues as desired and preferably comprise at least about 14, even more preferably about 25, and most preferably at most about 35 amino acid residues of antigen 5. Each region of such peptide preferably comprises up to 20 amino acid residues in length, more preferably up

to 15 residues in length and most preferably up to 10 amino acid residues in length as increases in length of a region may result in difficulty in peptide synthesis as well as retention of an undesirable property (e.g. , immunoglobulin binding or enzymatic activity) due to maintenance of conformational similarity between the peptide and the protein allergen from which it is derived. If desired, the amino acid sequences of the regions can be produced and joined by a linker to increase sensitivity to processing by antigen- presenting cells. Such a linker can be any non-epitope amino acid sequence or other appropriate linking or joining agent. To obtain preferred peptides comprising at least two regions, each comprising at least one T cell epitope, the regions are arranged in a configuration different from a naturally-occurring configuration of the regions in the allergen or a combination of different antigen 5s. For example, the regions containing T cell epitope(s) can be arranged in a contiguous configuration and can preferably be derived from the same antigen or a combination of antigen 5 homologues from different species. Noncontiguous is defined as an arrangement of regions containing T cell epitope(s) which is different from that of an amino acid sequence present in the protein allergen from which the regions are derived. Furthermore, the noncontiguous regions containing T cell epitopes can be arranged in a nonsequential order (e.g. , in an arrangement different from the order of the arrangement found in the native protein allergen from which the region containing T cell epitope(s) are derived).

The present invention further provides for modification or derivatization of peptides in a library. Modifications of peptides are well known to one of ordinary skill, and include phosphorylation, carboxymethylation, and acylation. Modifications may be effected by chemical or enzymatic means.

It is also possible to modify the structure of a peptide of the invention for such purposes as increasing solubility, enhancing therapeutic or preventive efficacy, or stability (e.g. , shelf life ex vivo, and resistance to proteolytic degradation in vivo). A modified peptide can , ^e produced in which the amino acid sequence has been altered, such as by amino acid substitution, deletion, or addition, to modify immunogenicity and/or reduce allergenicity, or to which a component has been added for the same purpose.

For example, a peptide can be modified so that it maintains the ability to induce T cell anergy and bind MHC proteins without the ability to induce a strong proliferative response

or possibly any proliferative response when administered in immunogenic form. In this instance, critical binding residues for the T cell receptor can be determined using known techniques (e.g. , substitution of each residue and determination of the presence or absence of T cell reactivity). Those residues shown to be essential to interact with the T cell receptor can be modified by replacing the essential amino acid with another, preferably similar amino acid residue (a conservative substitution) whose presence is shown to enhance, diminish but not eliminate, or not affect T cell reactivity. In addition, those amino acid residues that are not essential for T cell receptor interaction can be modified by being replaced by another amino acid whose incorporation may enhance, diminish or not affect T cell reactivity, but does not eliminate binding to relevant MHC.

Additionally, peptides of the invention can be modified by replacing an amino acid shown to be essential to interact with the MHC protein complex with another, preferably similar amino acid residue (conservative substitution) whose presence is shown to enhance, diminish but not eliminate, or not effect T cell activity. In addition, amino acid residues which are not essential for interaction with the MHC protein complex but which still bind the MHC protein complex can be modified by being replaced by another amino acid whose incorporation may enhance, not effect, or diminish but not eliminate T cell reactivity. Preferred amino acid substitutions for non-essential amino acids include, but are not limited to, substitutions with alanine, glutamic acid, or a methyl amino acid.

Another example of a modification of peptides is substitution of cysteine residues preferably with serine, threonine, leucine or glutamic acid to minimize dimerization via disulfide linkages.

In order to enhance stability and/or reactivity, peptides can also be modified to incorporate one or more polymorphism in the amino acid sequence of a protein allergen resulting from natural allelic variation or from homologous peptides. Additionally, D-amino acids, non- natural amino acids, or non-amino acid analogs can be substituted or added to produce a modified peptide within the scope of this invention. Furthermore, peptides of the present invention can be modified using the polyethylene glycol (PEG) method of A. Sehon and co-workers [Wie et al. , supra] to produce a peptide conjugated with PEG. In addition, PEG can be added during chemical synthesis of a peptide of the invention. Modifications of peptides or portions thereof can also include reduction/alkylation [Tarr in: Methods of

Protein Microcharacterizatwn, J E Silver ed , Humana Press Clifton, NJ, pp 155-194) (1986)], acylation [Tarr, supra], esterification [Tarr, supra], chemical coupling to an appropriate carrier [Mishell and Shngi, eds , Selected Methods in Cellular Immunology , WH Freeman San Francisco, CA (1980), U S Patent 4,939,239], or mild formalin treatment [Marsh, Int Arch Allergy App Immunol , 42 199-215 (1971)]

To facilitate purification and potentially increase solubility of peptides of the invention, it is possible to add reporter group(s) to the peptide backbone For example, polyhistidine can be added to a peptide to purify the peptide on immobilized metal ion affinity chromatography [Hochuli, E et al , Bio/Technology 6 1321-1325 (1988)] In addition, specific endoprotease cleavage sites can be introduced, if desired, between a reporter group and amino acid sequences of a peptide to facilitate isolation of peptides free of irrelevant sequences In order to successfully desensitize an individual to a protein antigen, it may be necessary to increase the solubility of a peptide by adding functional groups to the peptide or by not including hydrophobic T cell epitopes or regions containing hydrophobic epitopes in the peptides

To potentially aid proper antigen processing of T cell epitopes within a peptide, canonical protease sensitive sites can be recombinantly or synthetically engineered between regions, each comprising at least one T cell epitope For example, charged amino acid pairs, such as KK or RR, can be introduced between regions within a peptide during recombinant construction of the peptide The resulting peptide can be rendered sensitive to proteolytic cleavage, e g , with cathepsin and/or other trypsin-like enzymes, to generate portions of the peptide containing one or more T cell epitopes In addition, such charged ammo acid residues can result in an increase in solubility of a peptide

Fatty acyl peptide derivatives may also be prepared For example, and not by way of limitation, a free ammo group (N-terminal or lysyl) may be acylated, e g , myπstoylated In another embodiment an amino acid comprising an aliphatic side chain of the structure - (CH ₂ )„CH ₃ may be incorporated m peptides of the library This and other peptide-fatty acid conjugates suitable for use m the present invention are disclosed in U K Patent GB- 8809162 4, International Patent Application PCT/AU89/00166

Activity Assays With Peptides of the Invention Numerous assays are known in immunology for evaluating the lmmunomodulatory activity of an antigen For example, the immunodominant vespid venom can be used in diagnostic assays for allergic diseases, which are described in detail, infra In general, such peptides can be tested for the ability to induce proliferation of T cells from allergic subjects, without reacting with antibodies specific for antigen 5 Preferably, such antibodies that are not reactive with the peptides of the invention in the diagnostic assay are of the IgE class It is important to note that natural allergen-specific antibodies have been found to bind weakly to denatured vespid venom allergens

The peptides of the invention can be tested in a proliferation assay for T cell responses Generally, lymphocytes from a sensitized host are obtained The host can be a mouse that has been immunized with a vespid venom antigen 5, or with a crossreactive protein, such as testes specific protein Using techniques that are well known in the art, T lymphocyte response to the peptide can be measured in vitro In a specific embodiment, infra, T cell responses are detected by measuring incorporation of ³ H-thymιdme, which increases with DNA synthesis associated with proliferation Cell proliferation can also be detected using an MTT assay [Mossman, J Immunol Methods, 65 55-63 (1983), Niks and Otto, J Immunol Methods, 130 140-151 (1990)] Alternatively, lymphokine production assays can be practiced according to the present invention In one embodiment, lymphokine production can be assayed using lmmunological or co-stimulation assays [see, e g , Fehlner et al , J Immunol , 146 799 (1991)] or using the ELISPOT technique [Czerkmsky, et al , J Immunol Methods, 110 29 (1988)] Alternatively, mRNA for lymphokines can be detected, e g , by amplification [see Brenner, et al , Biotechniques , 7 1096 (1989)] or in situ hybridization [see, e g , Kasaian and Biron, J Immunol , 142 1287 (1989)] Of particular interest are those individuals whose T cells produce lymphokines associated with IgE isotype switch events, e g , IL-4 and IL-5 [Purkeson and Isakson, J Exp Med , 175 973-982 (1992)] Also of interest are the peptide fragments of the vespid venom antigen 5 that contain epitopes recognized by T cells involved in IgE switch events Any method for detecting T cell proliferation known in the art can be used with the immunodominant vespid antigen 5 peptides obtained according to the present invention

Thus, in a preferred aspect, the peptides of the present invention can be used in in vitro assays with peripheral blood lymphocytes or cell lines derived from peripheral blood lymphocytes, obtained from vespid venom antigen 5 sensitive individuals to detect secretion of lymphokines ordinarily associated with allergic responses, e.g. , IL-4. Such assays may indicate which epitopes are responsible or associated with the allergic condition. In this way, specific epitopes responsible for T cell responses associated with allergic response can be identified. The sequences of such epitopes can be compared to other vespid venom antigen 5s and to environmental or autologous proteins to determine if there are sequence similarities that suggest possible cross-reactivity. The peptides can be tested for the ability to induce T cell anergy, e.g. , by mega-dose administration, modification to produce an epitope antagonist, administration in the absence of the appropriate costimulatory signals, and other methods thought to result in T cell anergy. Peptides containing such epitopes are ideal candidates for therapeutics.

In a further embodiment, the polypeptides of the invention can be used directly in assays to detect the extent of cross-reactivity with other environmental proteins and/or homologous proteins, with which they share sequence similarity. In particular, the immunodominant fragments of the vespid venom antigen 5s that have sequence similarity with such environmental, and more particularly, homologous proteins can be evaluated for cross reactivity with antibodies or T cell specific for such proteins. In a specific embodiment, the cross reactivity of vespid venom antigen 5s with human and mouse testes specific protein can be evaluated.

Diagnostic and Therapeutic Uses of the Peptides The present invention identifies therapeutically relevant polypeptide fragment of vespid venom antigen 5. The invention contemplates use of the immunoreactive fragments of vespid venom antigen 5 for the preparation of diagnostic or therapeutic compositions, for the use in the diagnosis and therapy of vespid venom allergen-specific allergic conditions. In particular, vespid antigen 5 from Dolichovespula maculata (white-face hornet) (Doi V), Dolichovespula arenaria (yellow hornet) (Doi a V), Vespula vulgaris (yellowjacket) (Ves v V), Vespula maculifrons (yellowjacket) (Ves m V), Polistes annularis (wasp) (Pol a V), and Polistes exclamans (wasp) (Pol e V) are contemplated for use in diagnosis and therapy according to the present invention. Other vespid species known to harbor antigen 5, and thus represent additional antigen 5 species of the invention, include but are not

limited to Vespa crabo (European hornet), V. flavopilosa (yellow jacket), V. germanica (yellowjacket), V. pennsylvamca (yellowjacket), V. squamosa (yellowjacket), V. vidua (yellowjacket), and P. fuscatus (paperwasp).

Diagnostic Methods

The present invention contemplates in vitro diagnostic assays on peripheral blood lymphocytes, as described supra. Such diagnostic assays can give detailed information about the antigen 5-specific T cell responses, the phenotype of the T cell response, and preferably the T cell epitope of the antigen 5 involved in T cell responses. The immunodominant epitope and the epitope involved in IgE isotype class switch events can be detected, if they are not the same. In particular, the T cell epitopes of vespid venom antigen 5s that stimulate proliferation and/or lymphokine secretion of T cells of a phenotype associated with IgE isotype class switching events can be identified for a specific individual, or for a class of individuals who share MHC haplotype or a predominant T cell receptor variable region expression, or both.

In vivo assays for allergenicity generally consist of skin prick sensitivity assays, in which serially diluted amounts of an allergen are administered either subcutaneously or intradermally into a patient's skin, and wheel and erythema reactions are detected. As with in vitro assays, the availability of pure venom antigen 5 peptides greatly increases the value of the results of the in vivo diagnostic assays since cross-reactivity with impurities in extracts prepared from vespid venom sacs, and uncertainty about the epitope specificity can be avoided.

Therapeutic Methods

Therapeutic compositions of the invention (see, infra) can be used in immunotherapy, also referred to as hyposensitization therapy. Immunotherapy has proven effective in allergic diseases, particular insect allergy. Allergens are administered parenterally over a long period of time in gradually increasing doses. Such therapy may be particularly effective when the allergen or allergens to which the patient is sensitive have been specifically identified and the therapy is targeted to those allergen(s). Thus, the availability of specific, pure vespid venom antigen 5 peptide(s) in large quantities is important for immunotherapy of allergy.

The present invention contemplates use of peptides containing at least an lmmunomodulatory T cell epitope of a vespid venom antigen 5 to induce specific T cell anergy to the vespid venom antigen 5 Such lmmunomodulatory peptide contains one or more immunodominant epitope of antigen 5 A peptide comprising such a T cell epitope and lacking a B cell epitope can be administered to a patient As discussed supra, 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 peptides can be injected subcutaneously to decrease the T cell response to the entire molecule, e g , as described by Bπner et al [Proc Natl Acad Sci U S A , 90 7608-12 (1993)] In another embodiment, one or more peptide can be administered intranasally to suppress allergen-specific responses in naive and sensitized subjects [see e g , Hoyne et al , J Exp Med , 178 1783-88 (1993)]

Administration of a vespid venom antigen peptide of the invention is expected to induce anergy, resulting in cessation of allergen-specific antibody production or allergen-specific T cell response, or both, and thus, have a therapeutic effect

In a preferred aspect of the invention, peptide-based therapy to induce T cell anergy is customized for each individual or a group of individuals Using the diagnostic methods of the present invention, the specific T cell epitope or epitopes of a vespid venom antigen 5 involved m the allergic response can be identified Peptides comprising these epitopes can then be used in an individualized immunotherapy regimen

For example, by analogy and based on the data shown in Table 4, infra, a treatment protocol for BALB/c mice could include peptides 1 , 5, 6. 1 1 , 15, 20, and 18, while tre , nent of ASW/Sn mice might include peptides 6, 7, and 18, treatment of C3H/He might include peptides 2, 5, and 15, and treatment of P/J mice might include peptides 1 , 9, and 15

Administration of the therapeutic compositions of the present invention to desensitize an individual can be carried out using known techniques For example, one or more

immunodominant peptides of the invention comprising at least one T cell epitope can be administered in combination with an appropriate diluent, a carrier, and/or an adjuvant To induce T cell anergy in an individual, the therapeutic composition is preferably administered in non-immunogenic form, e g , it does not contain adjuvant Pharmaceutically acceptable diluents include saline and aqueous buffer solutions

Pharmaceutically acceptable carriers include polyethylene glycol [Wie et al , International Archives of Allergy and Applied Immunology, 64 84-99 (1981)] and liposomes [Strejan et al , Journal of Neuroimmunology , 7 27 (1984)] Such compositions will generally be administered by injection (subcutaneous, intravenous, etc ), oral administration (e g , as in the form of a capsule), transdermal application, or transmucosal administration (e g nasal, buccal, or rectal administration) The therapeutic compositions of the invention are administered to individuals sensitive to vespid venom at dosages and for lengths of time effective to reduce sensitivity e , reduce the allergic response) of the individual to vespid stings A therapeutically effective amount of one or more of the same or different therapeutic compositions can be administered simultaneously or sequentially to an individual sensitive to vespid venom Effective amounts of the therapeutic compositions will vary according to factors such as the degree of sensitivity of the individual to vespid venom, the age, sex, and weight of the individual, and the ability of the peptide to stimulate a T cell response m the individual

In yet another aspect of the present invention, a composition is provided comprising at least two immunodominant peptides (e g , a physical mixture of at least two peptides), each comprising at least one T cell epitope of antigen 5 The peptides are derived from the same or from different homologues of antigen 5 Such compositions can be administered in the form a therapeutic composition with a pharmaceutically acceptable carrier of diluent A therapeutically effective amount of one or more of such compositions can be administered simultaneously or sequentially to an individual sensitive to vespid venom

Transmucosal administration. According to the invention, an> transmucosal route of administration, including but not limited to nasal, rectal, oral, vaginal, buccal, etc can be employed It can be readily appreciated that any of the transmucosal routes of administration may be enhanced b> use of a mucosal penetration enhancer The term "mucosal penetration enhancer" refers to a reagent that increases the rate or facility of

transmucosal penetration of peptide, such as but not limited to, a bile salt, fatty acid, surfactant or alcohol In specific embodiments, the permeation enhancer can be sodium cholate, sodium dodecyl sulphate, sodium deoxycholate, taurodeoxycholate, sodium glycocholate, dimethylsulfoxide or ethanol Suitable penetration enhancers also include glycyrrhetmic acid [U S Patent No 5, 1 12,804 to Kowarski] and polysorbate-80, the latter preferably in combination with an non-ionic surfactant such as nonoxynol-9. laureth-9, poloxamer-124, octoxynol-9, or lauramide-DEA [European Patent EP 0 242 643 Bl by Stoltz] The selection of a particular mucosal penetration enhancer may depend on the characteristics of the specific mucosa These factors are addressed in greater detail below

Nasal Administration Transnasal or intranasal (nasal) delivery of an antigen 5 peptide can be used to suppress an allergen-specific response [see Hoyne et al , J Exp Med , 178 1783-1788 (1993)] Nasal delivery allows the passage of the protein to the blood stream directly after administering the product to the nose, without the necessity for deposition of the product in the lung Formulations for nasal delivery include those with dextran or cyclodextran For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached In one embodiment, the metered dose is delivered by drawing the peptide solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed The chamber is compressed to administer the solution In a specific embodiment, the chamber is a piston arrangement Such devices are commercially available Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed The opening is usually found m the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation Preferably, the nasal applicator will provide a metered amount of the nasal formulation, for administration of a measured dose of the peptιde(s)

Administration Via Suppositories In another aspect, peptide is formulated in a matrix suitable for rectal (or vaginal) insertion, / e , in a suppository The invention is not limited to any particular suppository formulation Indeed, many suppository formulations are known in the art, e g, as described in Remington 's Pharmaceutical Sciences, Physicia 's Desk Reference, and U S Pharmacopeia

Administration Via a Buccal Patch According to the invention, peptide can be formulated in a buccal patch for administration via the interior of the cheek It may be appreciated that a buccal patch constitutes another form of transmucosal administration The technology for preparing buccal patch formulations is known in the art, e g , Remington 's Pharmaceutical Sciences, supra

Oral-Pharyngeal Administration In yet another embodiment, peptide can be formulated for oral-pharyngeal, including sublingual and transbuccal, administration For example, peptide can be incorporated in a "candy" matrix, such as that described in U S Patent No 4,671 ,953, in a gum base, or a lozenge In another embodiment, the peptide can be formulated in a capsule or pill form for sublingual placement It is particularly contemplated that peptide for oral-pharyngeal administration may be formulated with a flavor masking agent or coating Many flavor masking agents for use with oral pharmaceuticals are known in the art, and can be selected for use with the present invention

Oral Administration In still a further embodiment, peptide can be formulated for oral administration via the stomach and intestinal mucosa For oral administration, peptide can be administered in a carrier designed for drug release in either the stomach (an acidic environment), or the intestines, or both Many capsules, pills, and matrices for oral administration of a drug are known in the art, and can be selected on the basis of compatibility with peptide, and the desired point and rate of drug release by the ordinary skilled physician Oral administration of peptide may require higher dosages than other routes of administration to overcome the effects of first pass metabolism by the liver

Transdermal Administration In a further embodiment, as noted above, the present invention is directed to transdermal administration of peptide Various and numerous methods are known in the art for transdermal administration of a compound, e g , via a transdermal patch These methods and associated devices provide for control of the rate and quantity of administration of a drug, and some allow for continuous modulation of drug deliver) Transdermal patches are described in, for example, U S Patent No 5,407,713, issued April 18, 1995 to Rolando et al , U S Patent No 5,352,456, issued October 4, 1004 to Fallon et al , U S Patent No 5,332,213 issued August 9, 1994 to D'Angelo et al , U S Patent No 5,336, 168, issued August 9, 1994 to Sibahs, U S

Patent No. 5,290,561 , issued March 1 , 1994 to Farhadieh et al. ; U.S. Patent No. 5,254,346, issued October 19, 1993 to Tucker et al. ; U.S. Patent No. 5.164.189, issued November 17, 1992 to Berger et al. ; U.S. Patent No. 5, 163,899, issued November 17, 1992 to Sibalis; U.S. Patent Nos. 5,088,977 and 5,087,240, both issued February 18, 1992 to Sibalis; U.S. Patent No. 5,008, 1 10, issued April 16, 1991 to Benecke et al. ; and U.S. Patent No. 4,921 ,475, issued May 1 , 1990 to Sibalis. the disclosure of each of which is incorporated herein by reference in its entirety.

It can be readily appreciated that a transdermal route of administration may be enhanced by use of a dermal penetration enhancer, e.g. , such as enhancers described in U.S. Patent No. 5,164,189 (supra), U.S. Patent No. 5,008,110 (supra), and U.S. Patent No. 4,879, 119, issued November 7, 1989 to Aruga et al.. the disclosure of each of which is incorporated herein by reference in its entirety.

Pharmaceutically Acceptable Compositions

The in vivo diagnostic or therapeutic compositions of the invention may also contain appropriate pharmaceutically acceptable carriers, excipients, diluents and adjuvants. As used herein, the term "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, although a pharmaceutically acceptable carrier may share the attributes of such approved or recognized carriers without having itself been approved or recognized. Examples of suitable pharmaceutical carriers are provided 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 preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include mannitol, human serum albumin (HSA), starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium carbonate, magnesium stearate, sodium stearate, glycerol monostearate,

talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like These compositions can take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained-release formulations and the like

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 MAPPING T CELL EPITOPES OF POL M Ae5

In the present Example, a series of 15 to 20-resιdue peptides with 10-resιdue overlaps to encompass the entire white faced hornet Ag5 molecule (form 2) was synthesized The sequences of these peptides are given in Figure 2 These peptides were used for mapping T cell epitopes of hornet Ag5 by their stimulation of specific muπne spleen cells immunized with native or recombinant Antigen 5 These peptides were also used to study the cross reacting T cell epitopes of hornet antigen 5 and a mouse testis protein Tpx [Mizuku et al , Mammalian Genome, 3 274-280 ( 1992)] This cross reactivity is suggested by the partial sequence identity of hornet antigen 5 with mouse and human testis proteins (Figure 3)

Results Antibody response in mice of different haplotypes to recombinant hornet Ag5 Five strains of mice were immunized with recombinant (r) white faced hornet Ag5 and alum as adjuvant Four strains were found to be high antibody responders for r-white faced hornet Ag5 BALB/v byj, ASW/Sn, C3H/He, and P/J Antibody responses were evaluated by direct ELISA [King et al , J Immunol . 154 577 (1995)] The antibody titers (the reciprocal of the dilution yielding 50% of maximum signal extrapolated from ELISA curves in wells coated with l-Dol m 5 02 or n-Dol m 5 01 ) of these sera for n-(natural)

and r-Ag5 are given in Table 3. ^" One strain, C57B1/6, was found to be a poor antibody responder.

Table 3

Antibody for Mice of Different Strains Immunized with Recombinant White faced hornet Ag5

Mice Half maximal Ab t ter*

*Sera are from week five bleeding after three intraperitoneal immunizations with 0.2 ml of 10 μg/ml r-Dol m 5.02 and alum (5 μg/ml) in 0.05 m sodium phosphate buffer, pH 6.2, on weeks 0, 2, and 4.

T cell response in BALB/c mice to natural hornet Ag5 and its recombinant protein or fragments. Figures 4 and 5 show the proliferative responses of spleen cells from BALB/c mice immunized 4 times with n- and r-Ag5 respectively. Nearly identical results show that r-Ag5 and n-Ag5 share common continuous T cell epitopes. Previous studies have shown that r-Ag5 lacks the discontinuous B cell epitopes of n-Ag5, as r-Ag5 is not properly folded with the disulfide bonds of n-Ag5 [King et al. , J. Immunol. , 154:577-584 (1995)]. Figure 6 shows the response of spleen cells from BALB/c mice immunized 5 times with r-Ag5. Higher proliferative responses were observed than those in Figure 5, where mice were immunized 4 times with r-Ag5. The results in Figure 4-6 together indicate that peptides 1, 5, 6, 11, 15, 20, and 18 contain the T cell epitopes of Ag5, if we assume that peptides have stimulation indices of > 4 represent positive responses with a high degree of certainty.

Figures 7 and 8 show the proliferation data with spleen cells from mice immunized 4 times with recombinant N-terminal and C-terminal fragments of antigen 5, labelled IN (residue 1-114) and C (residue 151-204), respectively. Similar but higher proliferation results were obtained with mice after 5 times immunization, as given in Figure 9 and 10. As to be expected, spleen cells from r-fragment IN or C immunized mice were stimulated by peptides whose sequences are represented by the immunogen. There is one exception

shown in Figure 9 spleen cells from r-fragment IN (residue 1-114) immunized mice were stimulated by peptide 18 (residue 176-195)

T cell response in mice of different haplotypes to recombinant hornet Ag5 Figures 11 to 13 show the proliferative response of spleen cells for mice of ASW/Sn, P/J, and C3H/He, respectively The results for ASW/Sn mice in Figure 1 1 indicate that there are four peptides with stimulation indices of >4 peptides 6, 7, 9, and 18 The results for P/J mice in Figure 12 show multiple peptides with stimulation indices of >4 peptides 1 , 2, 5, 9, 12, 13, 14, 15, 20, and 17 The data for C3H/He mice in Figure 12 indicate three peptides with stimulation indices of >4 peptides 2, 5 and 15

Figure 14 shows the proliferation responses of spleen cells for C57B1/6 mice Several peptides had stimulation indices of > 2, but only one peptide, number 9, showed a stimulation index approaching 4 A lower proliferation response compared to the othei mouse strains was also observed with r-Ag5 as a control These data indicate that

C57B1/6 mice showed poor T cell responses to white faced hornet Ag5, as contrasted to the other 4 strains of mice tested (Table 4)

Table 4

Summary of proliferation indices of r-Ag5 specific spleen cells from mice of different strains on stimulation with the dominant T cell epitope peptides ¹

Blank 3970 cpm 5640 cpm 3130 cpm 1730 cpm 1050 cpm

NOTES

1. Dominant T cell epitopes are defined as those peptides which showed stimulation indices of >4 in at least one of 5 strains of mice tested. 2. Peptides in bold face represent those with stimulation indices of >4 in 2 or more strains of mice. 3. Stimulation index values are taken from Figure 5, 9, 10, 11 and 12, in which spleen cells were from mice immunized 5 times with r-Ag5. The exceptions are those values in parentheses which ar taken from Figure 4 in which spleen cells were from 4 times r-Ag5 immunized mice.

T cell epitopes of hornet Ag5 and a mouse testis protein Tpx. Cross reactivity of these two proteins was studied in mice immunized with their fragments (Figures 15 and 16). Spleen cells from BALB/c mice immunized with r-fragment Tpx-N (residue 14-106) responded equally well to stimulation with the immunogen or the r-fragment IN of hornet Ag5 (residue 1-114). Similarly spleen cells from BALB/c mice immunized with r- fragment Tpx-C (residue 101-163) responded equally well to stimulation with the immunogen or the r-fragment C of hornet Ag5 (residue 151-204).

Proliferation of Tpx-N or -C primed spleen cells was also tested on stimulation with synthetic peptides which correspond to sequences present in r-fragment IN and C of hornet Ag5 The data in Figure 15 show high stimulation indices (greater than 6) for both r- fragments Tpx-N and fragment IN, only moderate stimulation indices (about 3) for peptides 7, 8 and 9, and near baseline stimulation indices of about 2 or less for the remaining peptides The data in Figure 16 show high stimulation indices (greater than 15) for Tpx-C, 9 for fragment C, 6 for peptide 18, and 2 or less for all other peptides

The data in Figure 15 and 16 were obtained from mice after 4 immunizations Further studies after five immunizations are planned as the data in Figures 7 to 10 indicate that stronger stimulation indices are obtained after longer immunization

Discussion The present Example defines indices of 4 or greater for proliferation of Ag5- specific cells as containing a T cell epitope of Ag5 With this definition, the data in Figures 3-13 for mice of 5 strains with different haplotypes would indicate that the T cell epitopes of Ag5 are distributed throughout the entire molecule This is summarized in Table 4 As C57B1/6 mice is a poor responder, its data are excluded in the following comparison

The data in Table 4 indicate that 14 peptides contain T cell epitopes of Ag5 Three of these peptides are recognized by three of the four strains tested, five of them by two strains and the remaining six by only one strain The peptides which are recognized by two or three strains of mice are indicated in bold face characters in Table 2 They are peptides 1. 2. 5, 6, 9, 15, 20 and 18 As can be seen in Figure 1 , there are extensive sequence identities of these white faced hornet peptides with those of the other vespids This is particularly apparent for peptides 6, 20, and 18

In addition to the seven vespid Ag5 sequences in Figure 1. there are eight other vespid Ag5s with known sequences They are nearly identical to those in Figure 1 , depending on their species group [Lu, et al , J Immunol , 150 2823-2830 (1993), Hoffman, J Allergy Clin Immunol , 92 707-716 (1993)] Fire ant venom allergen Sol I 3 also has sequence similarity with vespid Ag5s [Hoffman, J Allergy Clin Immunol . 91 71-78 (1993)] Peptides 9, 20 and 18 of white faced hornet Ag5 have a high degree of sequence identity with the corresponding peptides of Sol l 3

It is most likely that these vespid and fire ant Ag5s have T cell epitopes of identical and/or similar sequences These epitopes studies are being continued with yellowjacket or paper wasp Ag5 immunized mice

The finding of cross reactive T cell epitopes of hornet Ag5 and a mammalian testis protein is interesting, although its significance in clinical allergy, if any, is unknown Published reports do not indicate any unusual distribution of male and female insect allergic patients Our own unpublished studies showed that male and female BALB/c mice gave indistinguishable antibody response when immunized with hornet Ag5 in presence of alum

The present invention is not to be limited in scope by the specific embodiments describe 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

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description

Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties