# Patent

## Title
Methods and reagents for decreasing clinical reaction to allergy

## Abstract

The present invention provides methods and compositions for treating or preventing allergic reactions, particularly anaphylactic reactions. Methods of the present invention involve administering microorganisms to allergic subjects, where the microorganisms contain a recombinant version of the protein allergen. The recombinant version can be wild-type or may include mutations within IgE epitopes of the protein allergen. Preferably the compositions are administered rectally. Particularly preferred microorganisms are bacteria such as E. coli. Any allergen may be used in the inventive methods. Particularly preferred allergens are anaphylactic allergens including protein allergens found in foods, venoms, drugs and latex. The inventive compositions and methods are demonstrated in the treatment of peanut-induced anaphylaxis.

## Background
PRIORITY INFORMATION
The present application is a continuation of U.S. Ser. No. 10/899,551 filed Jul. 26, 2004, now abandoned which is a continuation-in-part of U.S. Ser. No. 09/731,375 filed Dec. 6, 2000 now U.S. Pat. No. 8,153,414 which claims the benefit of U.S. Ser. No. 60/195,035 filed Apr. 6, 2000. The present application is also a continuation-in-part of U.S. Ser. No. 10/100,303 filed Mar. 18, 2002 now abandoned. These and every other U.S. Patent Application cited herein are incorporated in their entirety by reference.


GOVERNMENT FUNDING
The United States government may have rights in this invention by virtue of grants AI-43668 and AI-01666 from the National Institute of Health.


In accordance with 37 C.F.R. §1.52(e)(5), a Sequence Listing in the form of a text file (entitled “Sequence_Listing.txt,” created on Dec. 2, 2009, and 36 kilobytes) is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Allergic disease is a common health problem affecting humans and companion animals (mainly dogs and cats) alike. Allergies exist to pollens, mites, animal danders or excretions, fungi, insects, foods, latex, drugs and other substances present in the environment. It is estimated that up to 8% of young children and 2% of adults have allergic reactions just to foods alone. Some allergic reactions (especially those to insects, foods, latex and drugs) can be so severe as to be life threatening.
Allergic reactions result when an individual's immune system overreacts, or reacts inappropriately, to an encountered allergen. Typically, there is no allergic reaction the first time an individual is exposed to a particular allergen. However, it is the initial response to an allergen that primes the system for subsequent allergic reactions. In particular, the allergen is taken up by antigen presenting cells (APCs; e.g., macrophages and dendritic cells) that degrade the allergen and then display allergen fragments to T-cells. T-cells, in particular CD4+ “helper” T-cells, respond by secreting a collection of cytokines that have effects on other immune system cells. The profile of cytokines secreted by responding CD4+ T-cells determines whether subsequent exposures to the allergen will induce allergic reactions. Two classes of CD4+ T-cells (Th1 and Th2; T-lymphocyte helper type 1 and 2) influence the type of immune response that is mounted against an allergen.
The Th1-type immune response involves the stimulation of cellular immunity to allergens and is characterized by the secretion of IL-2, IL-6, IL-12, IFN-γ and TNF-β by CD4+ T helper cells and the production of IgG antibodies. Exposure of CD4+ T-cells to allergens can also activate the cells to develop into Th2 cells, which secrete IL-4, IL-5, IL-10 and IL-13. One effect of IL-4 production is to stimulate the maturation of B-cells that produce IgE antibodies specific for the allergen. These allergen-specific IgE antibodies attach to receptors on the surface of mast cells and basophils, where they act as a trigger to initiate a rapid immune response to the next exposure to allergen. When the individual encounters the allergen a second time, the allergen is quickly bound by these surface-associated IgE molecules. Each allergen typically has more than one IgE binding site, so that the surface-bound IgE molecules quickly become crosslinked to one another through their simultaneous (direct or indirect) associations with allergen. Such cross-linking induces mast cell and basophil degranulation, resulting in the release of histamines and other substances that trigger allergic reactions. Individuals with high levels of IgE antibodies are known to be particularly prone to adverse allergic reactions.
The Th1- and Th2-type responses are antagonistic. In other words, one response inhibits secretions characterized by the other immune response. Thus, therapies to control the Th1- and Th2-mediated immune responses are highly desirable to control immune responses to allergens.
Other than avoidance and drugs (e.g., antihistamines, decongestants and steroids) that 1) only treat symptoms, 2) can have unfortunate side effects and 3) often only provide temporary relief, the only currently medically accepted treatment for allergies is immunotherapy. Immunotherapy involves the repeated injection of allergen extracts, over a period of years, to desensitize a patient to the allergen. Unfortunately, traditional immunotherapy is time consuming, usually involving years of treatment and often fails to achieve its goal of desensitizing the patient to the allergen. Furthermore, it is not the recommended treatment for anaphylactic allergens including food allergens (such as peanut allergens) due to the risk of anaphylaxis.
Noon first introduced allergen injection immunotherapy in 1911, a practice based primarily on empiricism with non-standardized extracts of variable quality (Noon, Lancet 1:1572, 1911). More recently the introduction of standardized extracts has made it possible to increase the efficacy of immunotherapy and double-blind placebo-controlled trials have demonstrated the efficacy of this form of therapy in allergic rhinitis, asthma and bee-sting hypersensitivity (BSAC Working Party, Clin. Exp. Allergy 23:1, 1993). However, an increased risk of anaphylaxis has accompanied this increased efficacy. For example, initial trials of immunotherapy to food allergens has demonstrated an unacceptable safety to efficacy ratio (Oppenheimer et al., J. Allergy Clin. Immun. 90:256, 1992; Sampson, J. Allergy Clin. Immun. 90:151, 1992; and Nelson et al., J. Allergy Clin. Immun. 99:744, 1996). Results like these have prompted investigators to seek alternative forms of immunotherapy as well as to seek other forms of treatment.
Initial trials with allergen-non-specific anti-IgE antibodies to deplete the patient of allergen-specific IgE antibodies have shown early promise (Boulet et al., American J. Respir. Crit. Care Med. 155:1835, 1997; Fahy et al., American J. Respir. Crit. Care Med. 155:1828, 1997; and Demoly and Bousquet American J. Resp. Crit. Care Med. 155:1825, 1997). On the other hand, trials utilizing immunogenic peptides that represent T-cell epitopes have been disappointing (Norman et al., J. Aller. Clin. Immunol. 99:S127, 1997). Another form of allergen-specific immunotherapy which utilizes injection of plasmid DNA remains unproven (Raz et al., Proc. Nat. Acad. Sci. USA 91:9519, 1994 and Hsu et al., Int. Immunol. 8:1405, 1996).
There remains a need for a safe and efficacious therapy for allergies, especially anaphylactic allergies such as food allergies where traditional immunotherapy is ill advised due to risk to the patient or lack of efficacy.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for treating or preventing allergic reactions, particularly anaphylactic reactions. Methods of the present invention involve administering microorganisms to allergic subjects, where the microorganisms contain a recombinant version of the protein allergen. The recombinant version can be wild-type or may include mutations within IgE epitopes of the protein allergen. Preferably the compositions are administered rectally. Particularly preferred microorganisms are bacteria such as E. coli. Any allergen may be used in the inventive methods. Particularly preferred allergens are anaphylactic allergens including protein allergens found in foods, venoms, drugs and latex. The inventive compositions and methods are demonstrated in the treatment of peanut-induced anaphylaxis.
ABBREVIATIONS
The following abbreviations are used throughout the application:
APC=antigen-presenting cell.
CPE=crude peanut extract.
CT=cholera toxin.
ig=intragastric gavage.
pr=per rectal.
sc=subcutaneous.
HKE=heat-killed E. coli. 
HKL=heat-killed L. monocytogenes. 
P123=a mixture of equal proportions of wild-type recombinant proteins Ara h 1, Ara h 2 and Ara h 3.
MP123=a mixture of equal proportions of mutant recombinant proteins Ara h 1, Ara h 2 and Ara h 3.
NP 12=a mixture of equal proportions of native proteins Ara h 1 and Ara h 2 that have been purified from crude peanut extract.
HKE-P123=a mixture of equal proportions of heat-killed E. coli cells expressing wild-type Ara h 1, Ara h 2 and Ara h 3.
HKE-MP123=a mixture of equal proportions of heat-killed E. coli cells expressing mutant Ara h 1, Ara h 2 and Ara h 3.
SPC=splenocyte.



BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 shows the nucleotide sequence of a cDNA clone of peanut allergen Ara h 1 (SEQ ID NO:1). This cDNA sequence has been deposited in GenBank under Accession No. L34402 (see also Burks et al., J. Clin. Invest. 96:1715, 1995).
FIG. 2 shows a full length native Ara h 1 amino acid sequence (SEQ ID NO:2). This amino acid sequence was predicted from the cDNA clone of SEQ ID NO:1.
FIG. 3 shows the nucleotide sequence of a cDNA clone of peanut allergen Ara h 2 (SEQ ID NO:3). This cDNA sequence has been deposited in GenBank under Accession No. L77197 (deposited by Stanley et al., 1996).
FIG. 4 shows a full length native Ara h 2 amino acid sequence (SEQ ID NO:4). Amino acids 1-3 of SEQ ID NO:4 are not encoded by the cDNA clone of SEQ ID NO:3. These amino acids were added based on a second cDNA clone of Ara h 2 that has been deposited in GenBank (Accession No. AY007229, see Viquez et al., J. Allergy Clin. Immunol. 107:713, 2001). Also, the C-terminal amino acid predicted from SEQ ID NO:3 is a tyrosine. However, sequencing of the native protein indicates that the C-terminal amino acid is an aspartic acid as shown in SEQ ID NO:4.
FIG. 5 shows the nucleotide sequence of a cDNA clone of peanut allergen Ara h 3 (SEQ ID NO:5). This cDNA sequence has been deposited in GenBank under Accession No. AF093541 (see also Rabjohn et al., J. Clin. Invest. 103:535, 1999).
FIG. 6 shows a full length native Ara h 3 amino acid sequence (SEQ ID NO:6). Amino acids 110, 111, 116, 117, 129, 202 and 290 of SEQ ID NO:6 are not encoded by SEQ ID NO:5. These amino acids have been amended after some sequencing errors were noted in the original published cDNA clone sequence. The signal peptide and amino acids 21-23 of SEQ ID NO:6 are not encoded by SEQ ID NO:5. These amino acids have been added based on a second cDNA clone of Ara h 3 that has been deposited in TrEMBL (Accession No. Q9SQH7, see Kleber-Janke et al., Int. Arch. Allergy Immunol. 119:265, 1999). A slightly different signal peptide is also present within a third cDNA clone of Ara h 3 that has also been deposited in TrEMBL (Accession No. Q8LKN1, deposited by Viquez et al., 2002).
FIG. 7 shows the amino acid sequence of an inventive wild-type recombinant Ara h 1 allergen (SEQ ID NO:53). The numbering is based on that of the full length native Ara h 1 amino acid sequence (see FIG. 2 and SEQ ID NO:2).
FIG. 8 shows the amino acid sequence of an inventive wild-type recombinant Ara h 2 allergen (SEQ ID NO:56). The numbering is based on that of the full length native Ara h 2 amino acid sequence (see FIG. 4 and SEQ ID NO:4).
FIG. 9 shows the amino acid sequence of an inventive wild-type recombinant Ara h 3 allergen (SEQ ID NO:58). The numbering is based on that of the full length native Ara h 3 amino acid sequence (see FIG. 6 and SEQ ID NO:6). Amino acids 346-530 of SEQ ID NO:6 (the C-terminal region) were not included in this wild-type recombinant Ara h 3 since this regions lacks linear IgE epitopes.
FIGS. 10A-C are graphs of allergen-specific release levels obtained with the cell-based mediator release assay of Example 7 as a function of cross-linking agent concentration (range: 0.001-1000 ng/ml).
FIG. 11 is an outline of the sensitization and challenge protocols that were used for the fourteen groups of mice (G1-G14) in the experiments of Example 10. Mice were first sensitized intraperitoneally with peanut over a 3 week period. The mice were then challenged intraperitoneally with low or high doses of various compositions 5 weeks after the initial sensitization.
FIG. 12 is an outline of the sensitization, desensitization and challenge protocols that were used for the ten groups of mice (G1-G10) in the experiments of Example 11. Mice were first sensitized intragastrically with peanut over an 8 week period. Mice were then treated with different compositions and via different routes 10-12 weeks after the initial sensitization. All mice were then challenged intragastrically with peanut 13 weeks after the initial sensitization.
FIG. 13 compares the average peanut-specific IgE levels at weeks 3, 8, 12, and 14 for the ten groups of mice (G1-G10) described in FIG. 12.
FIG. 14 compares the individual (symbols) and average (solid line) anaphylactic symptom scores that were determined after challenge for eight (G1-G8) of the ten groups of mice described in FIG. 12.
FIG. 15 compares the individual (symbols) and average (solid line) anaphylactic symptom scores that were determined after challenge for four (G1, and G8-G10) of the ten groups of mice described in FIG. 12.
FIG. 16 compares the individual (symbols) and average (solid line) body temperatures (° C.) that were determined after challenge for eight (G1-G8) of the ten groups of mice described in FIG. 12.
FIG. 17 compares the individual (symbols) and average (solid line) body temperatures (° C.) that were determined after challenge for four (G1, and G8-G10) of the ten groups of mice described in FIG. 12.
FIG. 18 compares the individual (symbols) and average (solid line) airway responses (peak expiratory flow in ml/min) that were determined after challenge for eight (G1-G8) of the ten groups of mice described in FIG. 12.
FIG. 19 compares the individual (symbols) and average (solid line) airway responses (peak expiratory flow in ml/min) that were determined after challenge for four (G1, and G8-G10) of the ten groups of mice described in FIG. 12.
FIG. 20 compares the plasma histamine concentrations (nM) that were determined after challenge for the ten groups of mice (G1-G10) described in FIG. 12.
FIG. 21 compares the plasma IL-4 concentrations (pg/ml) that were determined after challenge for the ten groups of mice (G1-G10) described in FIG. 12.
FIG. 22 compares the plasma IL-5 concentrations (pg/ml) that were determined after challenge for the ten groups of mice (G1-G10) described in FIG. 12.
FIG. 23 compares the plasma IFN-γ concentrations (pg/ml) that were determined after challenge for the ten groups of mice (G1-G10) described in FIG. 12.
FIG. 24 is an outline of the sensitization, desensitization and challenge protocols that were used for the six groups of mice (G1-G6) in the experiments of Example 12. Mice were sensitized intragastrically with peanut over an 8 week period. Mice were then treated with rectal administrations of different compositions 10-13 weeks after the initial sensitization. All mice were finally challenged intragastrically with peanut 14 weeks after the initial sensitization.
FIG. 25 compares the individual (symbols) and average (solid line) anaphylactic symptom scores that were determined after challenge for the six groups of mice (G1-G6) described in FIG. 24. The number of mice in each group were: G1 (sham)=17; G2 (HKE-MP123, 9 μg)=5; G3 (HKE-MP123, 90 μg)=12; G4 (MP123, 9 μg)=5; G5 (MP123, 90 μg)=5; G6 (naïve)=9. ***, p<0.001 vs. G1 (sham).
FIG. 26 compares the individual (symbols) and average (solid line) plasma histamine levels that were determined after challenge for the six groups of mice (G1-G6) described in FIG. 24. Blood was collected and plasma was obtained. Histamine levels were determined using an enzyme immunoassay kit. Data are mean±SEM for each group of mice. *, p<0.05 and ***, p<0.001 vs. G1 (sham). #, p<0.05 vs. G2 (HKE-MP123, 9 μg).
FIG. 27 compares average peanut specific IgE and IgG2a levels at weeks 3, 10, and 14 for the six groups of mice (G1-G6) described in FIG. 24. Sera from all groups of mice were obtained during sensitization (week 3), 1 day before desensitization (week 10) and 1 day before challenge (week 14). IgE levels (A) and IgG2a levels (B) were determined by ELISA. Data are mean±SEM for each group of mice. *, p<0.05 and ***, p<0.001 vs. G1 (sham). ##, p<0.01 and ###, p<0.001 vs. MP123.
FIG. 28 compares various cytokine levels that were determined from splenocyte (SPC) cultures taken after challenge for the six groups of mice (G1-G6) described in FIG. 24. Cell suspensions were cultured in complete culture medium in the presence (peanut) or absence (med) of CPE. Supernatants were collected 72 hours later, and cytokine levels were determined by ELISA. Results are expressed as mean±SEM of 2 duplicates of cultures (n=4). *, p<0.05; **, p<0.01 and ***, p<0.001 vs. G1 (sham). #, p<0.05 and ##, p<0.01 vs. G2 (HKE-MP123, 90 μg).
FIG. 29 shows histological images of sigmoid colon samples that were collected from peanut sensitized mice 24 hours after treatment (Panels A-C) or following peanut challenge at week 14 (Panels D-F) and fixed in formalin. Hematoxylin and eosin (H & E) stained sections showed normal histology. Panels A and D are from sham treated mice (G1). Panels B and E are from HKE-MP123, 90 μg treated mice (G2). Panels C and F are from naïve mice (G6).
FIG. 30 is an outline of the sensitization and challenge protocols that were used for the six groups of mice (G1-G6) in the experiments of Example 13. Mice were sensitized intragastrically with peanut over an 8 week period. Mice were then treated with rectal administrations of different compositions 10-12 weeks after the initial sensitization. Mice were challenged intragastrically 2, 6 and 10 weeks after the termination of therapy (week 14, 18, and 22 respectively post-desensitization). Following each challenge, 4 mice were sacrificed for collection of blood and tissue samples.
FIG. 31 compares the individual (symbols) and average (solid line) anaphylactic symptom scores that were determined after challenge for the six groups of mice (G1-G6) described in FIG. 30. Mice were challenged at week 14 (Panel A), 18 (Panel B) and 22 (Panel C). Anaphylactic symptom scores were determined 30 min following challenge. Bars indicate the median of 12 mice (Panel A), 8 mice (Panel B) and 4 mice (Panel C) in each group. *, p<0.05, and **, p<0.01 vs. G1 (sham).
FIG. 32 compares the individual (symbols) and average (solid line) plasma histamine levels that were determined after challenge for the six groups of mice (G1-G6) described in FIG. 30. Blood was collected and plasma was obtained. Histamine levels were measured using an enzyme immunoassay kit. Data are mean±SEM for each group of 12 mice (Panel A), 8 mice (Panel B) and 4 mice (Panel C). *, p<0.05, and **, p<0.01 vs. G1 (sham).
FIG. 33 compares average peanut specific IgE and IgG2a levels at weeks 3, 6, 8, 10, 14, 18 and 22 for the six groups of mice (G1-G6) described in FIG. 30. Sera from all groups of mice were obtained during sensitization (weeks 3, 6 and 8), before treatment (week 10), and one day prior to each challenge (weeks 14, 18 and 22). Peanut-specific IgE (A) and IgG2a (B) levels were determined by ELISA. Data are mean±SEM for each group. *, p<0.05 vs. G1 (sham); ***, p<0.001 vs. G1 (sham).
FIG. 34 compares various cytokine levels that were determined from splenocyte (SPC) cultures taken after challenge for the six groups of mice (G1-G6) described in FIG. 30. Cell suspensions were cultured in complete culture medium in the presence of CPE (peanut), or medium (medium) alone or Con A (data not shown). Supernatants were collected 72 hours later, and cytokine levels were determined by ELISA. Results are expressed as mean±SEM of 2 duplicate cultures of 4 mice. *, p<0.05; **, p<0.01 and ***, p<0.001 vs. G1 (sham).
FIG. 35 is a flowchart that outlines the first part of the clinical study of Example 14.
FIG. 36 is a flowchart that outlines the second part of the clinical study of Example 14.



DEFINITIONS
“Animal”: The term “animal”, as used herein, refers to humans as well as non-human animals, including, for example, mammals, birds, reptiles, amphibians and fish. Preferably, a non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate, or a pig). An animal may be a transgenic animal.
“Antigen”: The term “antigen”, as used herein, refers to a molecule that elicits production of an antibody (i.e., a humoral response) and/or an antigen-specific reaction with T-cells (i.e., a cellular response) in an animal.
“Allergen”: The term “allergen”, as used herein, refers to a subset of antigens which elicit the production of IgE antibodies. The allergens of the present invention are protein allergens. The Appendices describe a variety of known protein allergens that are encompassed by the present invention.
“Allergic reaction”: An “allergic reaction”, as defined herein, is an immune response that is IgE mediated with clinical symptoms primarily involving the cutaneous (e.g., uticana, angiodema, pruritus), respiratory (e.g., wheezing, coughing, laryngeal edema, rhinorrhea, watery/itching eyes), gastrointestinal (e.g., vomiting, abdominal pain, diarrhea) and cardiovascular (i.e., if a systemic reaction occurs) systems. For the purposes of the present invention, an asthmatic reaction is considered to be a form of allergic reaction.
“Anaphylactic allergen”: An “anaphylactic allergen”, as defined herein, belongs to a subset of allergens that are recognized to present a risk of anaphylactic reaction in allergic individuals when encountered in its natural state (e.g., within a food extract). For example, for the purposes of the present invention, pollen allergens, mite allergens, allergens in animal danders or excretions (e.g., saliva, urine) and fungi allergens are not generally considered to be anaphylactic allergens. On the other hand, food allergens, insect allergens and rubber allergens (e.g., from latex) are generally considered to be anaphylactic allergens. Food allergens are particularly preferred anaphylactic allergens for use in the practice of the present invention. In particular, nut and legume allergens (e.g., from peanut, walnut, almond, pecan, cashew, hazelnut, pistachio, pine nut, brazil nut), dairy allergens (e.g., from egg, milk), seed allergens (e.g., from sesame, poppy, mustard), soybean, wheat and seafood allergens (e.g., from shrimp, crab, lobster, clams, mussels, oysters, scallops, crayfish) are anaphylactic food allergens according to the present invention. Particularly interesting anaphylactic allergens are those to which reactions are commonly so severe as to create a risk of death.
“Anaphylaxis” or “anaphylactic reaction”: “Anaphylaxis” or an “anaphylactic reaction”, as defined herein, belong to a subset of allergic reactions characterized by mast cell degranulation secondary to cross-linking of the high-affinity IgE receptor on mast cells and basophils induced by an anaphylactic allergen with subsequent mediator release and the production of severe systemic pathological responses in target organs, e.g., airway, skin digestive tract and cardiovascular system. As is known in the art, the severity of an anaphylactic reaction may be monitored, for example, by assaying cutaneous reactions, puffiness around the eyes and mouth, vomiting and/or diarrhea, followed by respiratory reactions such as wheezing and labored respiration. The most severe anaphylactic reactions can result in loss of consciousness and/or death.
“Antigen presenting cell”: An “antigen presenting cell” or “APC”, as defined herein, is a cell which processes and presents antigens to T-cells to elicit an antigen-specific response, e.g., macrophages and dendritic cells.
“Associated with”: When two entities are “associated with” one another as described herein, they are linked by a direct or indirect covalent or non-covalent interaction. Indirect interactions might involve a third entity that is itself associated with both the first and second entities. Desirable non-covalent interactions include, for example, hydrogen bonding, van der Walls interactions, hydrophobic interactions, magnetic interactions, etc. In certain embodiments, the non-covalent interactions are ligand/receptor type interactions. Any ligand/receptor pair with a sufficient stability and specificity to operate in the context of the invention may be employed to associate two entities. To give but an example, a first entity may be covalently linked with biotin and a second entity with avidin. The strong non-covalent binding of biotin to avidin would then allow for association of the first entity with the second entity. In general, possible ligand/receptor pairs include antibody/antigen, protein/co-factor and enzyme/substrate pairs. Besides the commonly used biotin/avidin pair, these include without limitation, biotin/streptavidin, digoxigenin/anti-digoxigenin, FK506/FK506-binding protein (FKBP), rapamycin/FKBP, cyclophilin/cyclosporin and glutathione/glutathione transferase pairs. Other suitable ligand/receptor pairs would be recognized by those skilled in the art, e.g., monoclonal antibodies paired with a epitope tag such as, without limitation, glutathione-S-transferase (GST), c-myc, FLAG® and maltose binding protein (MBP) and further those described by Kessler pp. 105-152 of Advances in Mutagenesis” Ed. by Kessler, Springer-Verlag, 1990 and further those described in “Affinity Chromatography: Methods and Protocols (Methods in Molecular Biology)” Ed. by Pascal Baillon, Humana Press, 2000 and “Immobilized Affinity Techniques” by Hermanson et al, Academic Press, 1992.
“Epitope”: The term “epitope”, as used herein, refers to a binding site including an amino acid motif of between approximately six and fifteen amino acids which can be bound by an immunoglobulin (e.g., IgE, IgG, etc.) or recognized by a T-cell receptor when presented by an APC in conjunction with the major histocompatibility complex (MHC). A linear epitope is one where the amino acids are recognized in the context of a simple linear sequence. These linear epitopes are also commonly referred to as sequential epitopes in the art. A conformational epitope is one where the amino acids are recognized in the context of a particular three dimensional structure.
“Immunodominant epitope”: A particular epitope is considered to be “immunodominant” if it is (i) responsible for a significant fraction of the binding for a particular immunoglobulin type (e.g., IgE) observed with the native allergen and/or (ii) recognized by the particular immunoglobulin type in a significant fraction of sensitive individuals. An immunodominant epitope is often defined in reference to the other observed epitopes. For example, all IgE epitopes in a given allergen can be assayed simultaneously (e.g., by immunoblot) and the immunodominant epitopes can be identified by their strength as compared with the other epitopes. Usually, but not always, an immunodominant epitope will contribute at least 10% of the binding reactivity observed in such a study. Alternatively or additionally, an epitope can be classified as immunodominant if it is recognized by sera of a significant fraction, preferably at least a majority, more preferably at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, of sensitive individuals.
“Population”: The term “population”, as used herein, refers to human as well as non-human populations, including, for example, populations of mammals, birds, reptiles, amphibians and fish. Preferably, the non-humans are mammals (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, primates, or pigs). As used herein the terms “individual” or “subject” encompass any member of these populations.
“Wild-type recombinant allergen”: A “wild-type recombinant allergen”, as defined herein, is a protein that (a) includes substantially, and in certain embodiments exactly, the same amino acid sequence as a naturally occurring protein allergen and (b) was produced in a non-natural host of the protein allergen. In certain embodiments, a recombinant allergen is produced in culture, preferably in a unicellular host and more preferably in a bacterial host. In certain embodiments all immunodominant linear IgE epitopes within the protein allergen are preserved within a “wild-type” recombinant allergen. In certain embodiments, all non-immunodominant linear IgE epitopes are also preserved. In certain embodiments, a “wild-type” recombinant allergen may include the exact same amino acid sequence as a naturally occuring protein allergen. In other embodiments, a recombinant allergen may include substantially the same amino acid sequence. It is preferred that the “wild-type” recombinant allergen include an amino acid sequence that is at least 90%, 95%, or 99% identical to the sequence of the protein allergen. In particular, a “wild-type” recombinant allergen may include a small number of amino acid mutations outside of the linear IgE epitopes. Preferably these mutations are conservative substitutions. In certain embodiments, a recombinant allergen may include one or more terminal amino acids that are absent from the naturally occuring protein allergen. In particular, terminal amino acids may be added to increase expression of the recombinant allergen, as a consequence of the vector used for expression, etc. In addition, amino acid segments that are absent from the protein allergen may be added to the amino and/or carboxyl terminus of a recombinant allergen, e.g., tags for purification, labels for detection, tags that increase the solubility of the recombinant allergen, tags that increase the stability and/or bioavailability of the recombinant allergen, etc. A proteolytic cleavage site may be introduced at the junction of the added amino acid segment and the recombinant allergen terminus to enable removal of the added segment after the recombinant allergen has been purified, absorbed, etc. Common terminal modifications used in recombinant technology are described in Current Protocols in Molecular Biology Ed. by Ausubel et al., John Wiley & Sons, New York, N.Y., 1989 and Molecular Cloning: A Laboratory Manual Ed. by Sambrook et al., Cold Spring Harbor Press, Plainview, N.Y., 1989.
Further, it will be appreciated that the amino acid sequence of a protein allergen encountered by an APC in vivo (i.e., within an exposed animal) may, in certain cases, differ from the full length amino acid sequence that is encoded by a cDNA clone of the naturally occuring protein allergen. It is to be understood that the methods of the present invention encompass the preparation and testing of recombinant versions of these naturally occuring “non-full length” protein allergens. For example, in certain embodiments, a protein allergen may include a terminal signal peptide that is cleaved in the natural host after translation of the full length protein. In addition, in other embodiments APCs may encounter digestion fragments of the full length protein allergen. This is particularly the case for food allergens that must negotiate the acidic environment of the stomach and a variety of proteolytic enzymes on their journey from ingestion to absorption.
“Mutant recombinant allergen”: A “mutant recombinant allergen”, as defined herein, has the same properties as a “wild-type recombinant allergen” (defined above) expect that it further includes one or more mutations within one or more IgE epitopes. In certain preferred embodiments, the one or more mutations are located within one or more linear IgE epitopes of the naturally occuring allergen. Preferably the mutations reduce IgE binding to the one or more IgE epitopes.
“Reduced allergic (or anaphylactic) reaction”: A “reduced allergic (or anaphylactic) reaction”, as defined herein, involves a decrease in the clinical symptoms that are associated with exposure to an allergen (or anaphylactic allergen), when exposure occurs via the route through which an individual would naturally encounter the allergen (or anaphylactic allergen), e.g., via cutaneous, respiratory, gastrointestinal, ocular, nasal, aural, etc. exposure or via a subcutaneous injection (e.g., in the form of a bee sting) depending on the nature of the allergen (or anaphylactic allergen).
“Th1 response” and “Th2 response”: Certain preferred compositions of the present invention are characterized by their ability to suppress a Th2 response and/or to stimulate a Th1 response preferentially as compared with their ability to stimulate a Th2 response. Th1 and Th2 responses are well-established alternative immune system responses that are characterized by the production of different collections of cytokines and/or cofactors. For example, Th1 responses are generally associated with production of cytokines such as IL-1β, IL-2, IL-12, IL-18, IFN-α, IFN-γ, TNF-β, etc; Th2 responses are generally associated with the production of cytokines such as IL-4, IL-5, IL-10, etc. The extent of T-cell subset suppression or stimulation may be determined by any available means including, for example, intra-cytoplasmic cytokine determination. In preferred embodiments of the invention, Th2 suppression is assayed, for example, by quantitation of IL-4, IL-5, and/or IL-13 in stimulated T-cell culture supernatant or by assessment of T-cell intra-cytoplasmic (e.g., by protein staining or analysis of mRNA) IL-4, IL-5, and/or IL-13. Similarly, in preferred embodiments of the invention, Th1 stimulation is assayed, for example, by quantitation of IFN-α, IFN-γ, IL-2, IL-12, and/or IL-18 in activated T-cell culture supernatant or by assessment of intra-cytoplasmic levels of these cytokines.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION
The present application references various patents, patent applications and published references. The contents of each such reference are hereby incorporated by reference.
The present invention provides methods and compositions for treating or preventing allergic reactions. It is an aspect of the present invention that undesirable allergic reactions are treated or prevented by administering microorganisms that express recombinant allergens of interest. In certain preferred embodiments, the invention provides methods for treating anaphylaxis including anaphylactic reactions to food allergens. In some embodiments, IgE epitopes within the recombinant allergens are mutated to reduce binding to IgE antibodies. In certain embodiments the microorganisms are bacteria, preferably E. coli. The present invention encompasses the finding that subcutaneous and preferably rectal administration of the inventive compositions has a potent and persistent, therapeutic effect on allergy. Examples 1-14 describe the preparation and use of inventive compositions in the treatment of peanut-induced anaphylaxis in a mouse model. As described in detail below, peanut-induced anaphylaxis is the gold-standard of allergies—it is rarely outgrown and until the present invention there was no known treatment.
A. Host Microorganisms
Any microorganism capable of expressing recombinant allergens may be used as a delivery vehicle in accordance with the present invention. Such microorganisms include but are not limited to bacteria, viruses, fungi (including yeast), algae and protozoa. Bacteria are preferred, particularly bacteria such as E. coli that naturally colonize within humans, e.g., in the gastrointestinal tract.
Generally, microorganisms are single cell, single spore or single virion organisms. Microorganisms that can be genetically manipulated to produce a desired recombinant allergen are preferred (e.g., see Ausubel et al., Current Protocols in Molecular Biology. Wiley and Sons, Inc. 1999, incorporated herein by reference). Genetic manipulation includes mutation of the host genome, insertion of genetic material into the host genome, deletion of genetic material from the host genome, transformation of the host with extrachromosomal genetic material, transformation with linear plasmids, transformation with circular plasmids, insertion of genetic material into the host (e.g., injection of mRNA), insertion of transposons, and/or chemical modification of genetic material. Methods for constructing nucleic acids (including an expressible gene), and introducing such nucleic acids into an expression system to express the encoded protein are well established in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, incorporated herein by reference).
Some of the motivations for utilizing a microorganism for delivering recombinant allergens include (i) integrity of the delivery system prior to endocytosis and avoidance of accidental exposure to IgE antibodies, (ii) known mechanisms of endocytosis (often including targeting to particular cell types), (iii) ease of production of the delivered recombinant allergens, (iv) experimental accessibility of the organisms, including ease of genetic manipulation, (v) ability to guarantee release (e.g., by secretion) of the recombinant allergen after endocytosis, and (vi) the possibility that the encapsulating organism will also act as an adjuvant (e.g., L. monocytogenes, E. coli, etc.).
As demonstrated in the Examples, use of microorganisms such as bacteria as therapeutic delivery vehicles in accordance with the present invention offers many advantages over delivery of allergens that are not encapsulated inside microorganisms. First, encapsulation reduces exposure of the recombinant allergen to IgE antibodies and thereby provides protection from IgE-mediated allergic responses. Second, a range of microorganisms are known to act as adjuvants that downregulate Th2-type responses and/or upregulate Th1-type responses (for a review, see for example, Freytag et al. Curr Top Microbiol Immunol 236:215-36, 1999).
As noted, bacteria are preferred microorganisms of the present invention. Generally, bacteria are classified as gram-negative or gram-positive depending on the structure of the cell walls. Those skilled in the art are capable of identifying gram-negative and gram-positive bacteria which may be used to express recombinant allergens in accordance with the present invention. Non-limiting examples of genera and species of gram-negative bacteria include Escherichia coli, Vibro cholera, Salmonella, Listeria, Legionella, Shigella, Yersenia, Citrobacter, Enterobacter, Klebsiella, Morganella, Proteus, Providencia, Serratia, Plesiomonas, and Aeromonas. Non-limiting examples of genera and species of gram-positive bacteria which may be used in the present invention include Bacillus subtilis, Sporolactobacillus, Clostridium, Arthrobacter, Micrococcus, Mycobacterium, Peptococcus, Peptostreptococcus, and Lactococcus. 
Gram-negative bacterial systems for use as delivery vehicles are known and may be used in the present invention. For example, E. coli is a well-studied bacteria, and methods of protein expression in E. coli are well-established. Most strains of E. coli have the advantage of being non-pathogenic since E. coli is found naturally in the gut. Therefore, E. coli is preferred as a delivery vehicle in the present invention and was employed in the Examples. In addition, Calderwood et al. (U.S. Pat. No. 5,747,028) utilize Vibrio cholerae as a delivery vehicle for production of antigens for use as a live vaccine against infectious organisms. Miller and Mekalanos (U.S. Pat. No. 5,731,196) utilize Salmonella as delivery vehicle for production of antigens for use as a live vaccine against infectious organisms. Hess et al. (Proc. Natl. Acad. Sci. USA 93:1458-1463, 1996) utilize recombinant attenuated Salmonella which secretes antigenic determinants of Listeria as a live vaccine to protect against listeriosis. Donner et al. (WO 98/50067) utilize attenuated Salmonella typhimurium as a gram-negative host for secretion of polypeptides for controlling fertility and also teach that other attenuated gram-negative strains including Yersinia may be used to express and secrete such polypeptides. Gram-positive bacteria have also been studied as delivery vehicles for proteins (e.g., see WO 97/14806 that describes the use of Lactococcus).
In another preferred embodiment, yeast are used as protein delivery microorganisms. It is well known that yeast are amenable to genetic manipulation to express a protein or proteins of choice (Ausubel et al., supra). Furthermore, in general most yeast are non-pathogenic. Without limitation to these species, two well-characterized species of yeast are the budding yeast Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces pombe. Moreover, the administration of yeast that express protein antigens to alter an immune response has been studied (e.g., see U.S. Pat. No. 5,830,463).
Microorganisms of the present invention may be administered to a subject as live or dead microorganisms. Preferably if the microorganisms are administered as live microorganisms, they are non-pathogenic or attenuated pathogenic microorganisms. Use of non-pathogenic, attenuated and/or killed microorganisms reduces or eliminates toxicity which may be associated with the bacteria. For applications of the invention where live microorganisms are administered to individuals, preferably the microorganisms are attenuated and/or are administered in suitable encapsulation materials to decrease immune responses to the microorganism. Generally, attenuation involves genetically modifying the infectious pathogenic microorganism to reduce or eliminate the infectious ability of the microorganism. Preferably, the microorganism is attenuated such that an individual inoculated with the microorganism does not suffer any cytotoxic effects from the presence of the microorganism. Particularly preferred attenuated microorganisms are infectious intracellular pathogens which are phagocytosed by APCs in individuals who are exposed to the microorganism. Examples of microorganisms which are intracellular pathogens include Salmonella, Mycobacterium, Leishmania, Legionella, Listeria, and Shigella. 
In certain preferred embodiments, the microorganisms of the present invention are administered to subjects after killing the microorganisms. Any method of killing the microorganisms may be utilized that does not greatly alter the expressed polypeptides. Methods of killing microorganism include but are not limited to using heat, antibiotics, chemicals such as iodine, bleach, ozone, and alcohols, radioactivity (i.e., irradiation), UV light, electricity, and pressure. Preferred methods of killing microorganisms are reproducible and kill at least 99% of the microorganisms. Particularly preferred is the use of heat above 50° C. for a period of time that kills greater than 99% of the cells and preferably 100% of the cells.
B. Protein Allergens
In general, recombinant versions of any naturally occuring protein allergen may be expressed in a microorganism of the present invention. The recombinant versions can be “wild-type” or “mutant” versions of the natural allergen. Without limitation, preferred natural protein allergens are anaphylactic allergens, including those found in certain foods, venoms, drugs or rubber. Food allergens are particularly preferred. In particular, nut and legume allergens (e.g., from peanut, walnut, almond, pecan, cashew, hazelnut, pistachio, pine nut, brazil nut), dairy allergens (e.g., from egg, milk), seed allergens (e.g., from sesame, poppy, mustard), soybean, wheat and seafood allergens (e.g., from shrimp, crab, lobster, clams, mussels, oysters, scallops, crayfish) are preferred food allergens of the present invention.
The Appendices that are attached hereto present a representative and non-limiting list of certain known protein allergens that may be used in the present invention including numerous anaphylactic and food allergens. This list was adapted from the Danish Biotechnological Database (“BioBase”) which is maintained by the University of Aarhus, Denmark. As indicated, amino acid sequences are known for many of these proteins, either through knowledge of sequences of their cognate genes or through direct knowledge of protein sequences, or both. In addition, to date, over two thousand protein allergen sequences have been deposited in the protein and gene databases that are maintained by the National Center for Biotechnology Information (NCBI, Bethesda, Md.). Thus, it will be appreciated that a large number of naturally occuring allergens are known and that recombinant allergens corresponding to these are readily identifiable. It will also be appreciated that these recombinant allergens are readily expressed within inventive microorganisms. Methods for preparing recombinant proteins in microorganisms are well known in the art and are described in great detail in the Examples and further in Current Protocols in Molecular Biology Ed. by Ausubel et al., John Wiley & Sons, New York, N.Y., 1989 and Molecular Cloning: A Laboratory Manual Ed. by Sambrook et al., Cold Spring Harbor Press, Plainview, N.Y., 1989.
A variety of methods are also known for isolating, cloning and sequencing unknown protein allergens including, but not limited to, those methods described in the references cited in the Appendices; those described in reviews, e.g., Crameri, Allergy 56:S30, 2001; Appenzeller et al., Arch. Immunol. Ther. Exp. 49:19, 2001; Deviller, Allerg. Immunol. (Paris) 27:316, 1995; and Scheiner, Int. Arch. Allergy Immunol. 98:93, 1992; and those described in reference collections, e.g., Current Protocols in Molecular Biology Ed. by Ausubel et al., John Wiley & Sons, New York, N.Y., 1989 and Molecular Cloning: A Laboratory Manual Ed. by Sambrook et al., Cold Spring Harbor Press, Plainview, N.Y., 1989.
The amino acid sequence of a protein allergen encountered in vivo (i.e., within an exposed animal) may, in certain cases, differ from the full length amino acid sequence that is encoded by a cDNA clone of the natural allergen. The methods of the present invention encompass the use of microorganisms that express recombinant versions of these non-full length protein allergens. For example, in certain embodiments a protein allergen may include a signal peptide that is cleaved in the natural host after translation of the full length protein. In certain preferred embodiments of the present invention amino acid sequences predicted from cDNA clones may be compared with N-terminal and/or C-terminal sequences determined by amino acid sequencing of the isolated allergen. As is well known in the art, such comparisons allow post-translational modifications (e.g., signal peptide cleavages) to be identified and hence mature allergens to be fully characterized.
In addition, in other embodiments digestion fragments of the full length protein allergen may be encountered in vivo. This is particularly common for food allergens that must negotiate the acidic environment of the stomach and a variety of proteolytic enzymes on their journey from ingestion to absorption. Accordingly, in certain embodiments, it may prove advantageous to identify and characterize the amino acid sequence of an allergen or its fragments subsequent to processing within an animal. In certain embodiments, allergen fragments may be isolated from in vivo samples using standard purification techniques (e.g., samples taken from the blood, the gastrointestinal tract, etc. of an animal that has been exposed to the protein allergen). Alternatively, the fragments can be generated in vitro, e.g., by proteolytic digestion of a protein allergen by one or more gastric, pancreatic and intestinal proteases such as pepsin, parapepsin I and II, trypsin, chymotrypsin, elastase, carboxypeptidases, enterokinase, aminopeptidases and/or dipeptidases.
As noted above, in certain embodiments the recombinant allergens are “wild-type” versions of the natural allergen. In other embodiments the recombinant allergens are “mutant” versions of the natural allergen. Preferably the mutant recombinant allergens bind less IgE than the naturally occuring allergen. This is generally achieved by mutation of one or more IgE epitopes of the natural allergen as described in Examples 1-3 and in WO 97/24139, WO 99/38978, and WO 01/40264 each of which is incorporated herein by reference.
Briefly, the majority of natural occuring protein allergens include conformational and/or linear epitopes for immunoglobulins such as IgE. These have been identified for a large number of known allergens. For example, without limitation, IgE epitopes have been identified for allergens from the following foods: cow milk (Ball et al., Clin. Exp. Allergy 24:758, 1994), egg (Cooke and Sampson, J. Immunol. 159:2026, 1997), codfish (Aas and Elsayed, Dev. Biol. Stand. 29:90, 1975), hazel nut (Elsayed et al., Int. Arch. Allergy Appl. Immunol. 89:410, 1989), peanut (Burks et al., Eur. J. Biochemistry 245:334, 1997 and Stanley et al., Arch. Biochem. Biophys. 342:244, 1997), soybean (Herein et al., Int. Arch. Allergy Appl. Immunol. 92:193, 1990), and shrimp (Shanty et al., J. Immunol. 151:5354, 1993).
A variety of methods are also known in the art that can be used to identify the amino acids involved in conformational and/or linear epitopes (e.g., see Benjamin et al., Ann. Rev. Immunol. 2:67, 1984; Atassi, Eur. J. Biochem. 145:1, 1984; Getzoff et al., Adv. Immunol. 43:1, 1988; Jemmerson and Paterson, Biotechniques 4:18, 1986; Geysen et al., J. Immunol. Methods 102:259, 1987; see also, Current Protocols in Immunology Ed. by Coligan et al., John Wiley & Sons, New York, N.Y., 1991).
For example, conformational epitopes can be determined using phage display libraries (see, for example, Eichler and Houghten, Molecular Medicine Today 1:174, 1995 and Jensen-Jarolim et al., J. Appl. Clin. Immunol. 101:5153a, 1997) and by cross-linking antibodies to whole protein or protein fragments, typically antibodies obtained from a pooled patient population known to be allergic to the natural allergen. Once some or all of the conformational IgE epitopes are known, it is possible to modify one or more of the amino acids that comprise the epitope(s), using site directed mutagenesis by any of a number of techniques.
Similarly, linear epitopes can be determined using a technique commonly referred to as “scanning” (see Geysen et al., 1987, supra). As described in greater detail in Examples 1-3, the approach uses collections of overlapping peptides that span the entire length of the allergen. The peptides may be chosen such that they span the length of the amino acid sequence predicted from a cDNA clone; the length of the mature protein (i.e., including any post-translational modifications); or the length of an allergen fragment (e.g., a digestion resistant fragment). The approximate location of linear epitopes within a given amino acid sequence can, for example, be determined using peptides that are 6-15 amino acids in length and offset by 1-5 residues. It is to be understood that peptides having any length and offset may be used according to the present invention; however, the use of longer peptides decreases the resolution of individual epitopes and the use of shorter peptides increases the risk of missing an epitope. For long amino acid sequences, where cost of peptide synthesis is a major consideration, longer peptides and offsets are preferred. For example, peptides that include a linear IgE epitope are identified using a standard immunoassay with serum IgE taken from an individual or a pool of individuals that are known to be allergic to the allergen. It will be recognized that different individuals may generate IgE that recognize different epitopes on the same allergen. Thus, it is typically desirable to expose the peptides to a representative pool of serum samples, e.g., taken from at least 5-10, preferably at least 15, individuals with demonstrated allergy to the allergen. Comparing binding between individual sera is also advantageous since it allows immunodominant epitopes to be identified. Once peptides that include a linear IgE epitope have been identified, the specific amino acids that are involved in each of the linear IgE epitopes can be determined by repeating the process using different sets of shorter overlapping peptides that span the length of these peptides.
In preferred embodiments, once the specific amino acids that are involved in each of the linear IgE epitopes have been identified, sets of peptides that cover each linear IgE epitope are prepared that each include a single mutation (e.g., substitution, deletion or addition). As described in detail in Examples 1-3, these mutated peptides can be used to identify those amino acids that are most important for IgE binding and hence cause the largest reduction in IgE binding when mutated. Identification of these amino acid positions facilitates the preparation of mutated recombinant allergens with reduced IgE binding.
In preferred embodiments, the mutant recombinant allergen includes one or more mutations that disrupt one or more of the IgE epitopes. It is to be understood that the mutations may involve substitutions for any other amino acid and that the methods are in no way limited to substitutions with alanine or methioinine residues as described in the Examples. Additionally or alternatively, the mutations may involve one or more deletions within one or more IgE epitopes. Typically linear IgE epitopes are about 6 to about 10 amino acids in length. As shown in Examples 1-3, single mutations within these linear epitopes can dramatically reduce IgE binding. In certain embodiments 2, 3, 4, 5, 6 or more amino acids can be mutated within a linear IgE epitope of a mutant recombinant allergen.
In some embodiments, the mutant recombinant allergen retains the ability to activate T-cells and/or the capacity to bind IgG. As is well known in the art, the methods described above can also be used to detect IgG epitopes. T-cell epitopes can also be detected in this manner using, for example, a T-cell proliferation assay. In certain embodiments, it will be advantageous to compare the locations of IgE, IgG, and T-cell epitopes within the sequence of a natural allergen of interest. According to such embodiments, mutations within regions of overlap between IgE and IgG and/or T-cell epitopes are preferably avoided.
C. Inducible Systems
In certain embodiments of the invention, expression of recombinant allergens by a microorganism is regulated so that synthesis occurs at a controlled time after a live microorganism has been administered to a subject. Preferably induction of protein synthesis is regulated so that activation occurs after the microorganism is taken up by APCs and phagocytosed into the endosome. A desirable result of this regulation is that production of the allergen of interest occurs inside the APCs and therefore reduces or eliminates the exposure of the allergen to IgE molecules bound to the surface of histamine-releasing mast cells and basophils. This further reduces or eliminates the risk of anaphylaxis during administration of microorganisms that produce anaphylactic allergens.
Any method of controlling protein synthesis in the microorganism may be used in accordance with the present invention. Preferably, the method of controlling protein synthesis utilizes an inducible promoter operatively-linked to the gene of interest (e.g., a gene which encodes a signal peptide and recombinant allergen). Many systems for controlling transcription of a gene using an inducible promoter are known (e.g., see Ausubel et al. Current Protocols in Molecular Biology. Wiley and Sons. New York. 1999). Generally, inducible systems either utilize activation of the gene or derepression of the gene. It is preferred that the present invention utilizes activation of a gene to induce transcription. However, inducible systems using derepression of a gene may also be used in the present invention. Systems using activation are preferred because these systems are able to tightly control inactivation (and hence basal level synthesis) since derepression may result in low levels of transcription if the derepression is not tight.
Methods of inducing transcription include but are not limited to induction by the presence or absence of a chemical agent, induction using a nutrient starvation inducible promoter, induction using a phosphate starvation inducible promoter and induction using a temperature sensitive inducible promoter. A particularly preferred system for regulating gene expression utilizes tetracycline controllable expression system. Systems which utilize the tetracycline controllable expression system are commercially available (Clontech, Palo Alto, Calif.).
Another particularly preferred system for regulating gene expression utilizes an ecdysone-inducible expression system which is also commercially available (Invitrogen, Carlsbad, Calif.). The ecdysone-inducible expression system is based on the ability of ecdysone, an insect hormone, to activate gene expression by binding to the ecdysone receptor. The expression system utilizes a modified heterologous protein containing the ecdysone receptor, a viral trans activation domain (from VP16) and the retinoid X receptor derived from mammalian cells to bind to a modified ecdysone response element in the presence of a ligand such as ecdysone or an analog (e.g. muristerone A or ponasterone A).
It is preferred that inducible systems for use in the present invention utilize inducing agents that are non-toxic to mammalians cells including human cells. Furthermore, it is preferred that transcriptional inducing agents permeate cell membranes. More specifically for activation of protein synthesis in microorganisms after phagocytosis by APCs, transcriptional inducing agents must be able to pass through cell membranes of the APCs and cell membranes of the microorganism. Since both tetracycline and ecdysone are able to pass through cell membranes and are non-toxic, tetracycline-inducible systems and ecdysone-inducible systems are ideally suited for use in the present invention. However, the use of inducible systems in the present invention is not limited to those systems.
It is also preferred that microorganisms that have not been phagocytosed are killed before induction of genes expressing recombinant allergens of interest. A preferred method of killing bacteria is to use antibiotics which are not permeable to mammalian cell membranes such that only bacteria that are not phagocytosed are killed. Those having ordinary skill in the art are readily aware of antibiotics which may be used. Such antibiotics include but are not limited to penicillin, ampicillin, cephalosporin, griseofulvin, bacitracin, polymyxin b, amphotericin b, erythromycin, neomycin, streptomycin, tetracycline, vancomycin, gentamicin, and rifamycin. The use of antibiotics in accordance with the present embodiment reduces or eliminates the production of recombinant allergens by microorganisms outside APCs. It is preferable to reduce or eliminate exposure of allergen-producing microorganisms to the immune system, especially microorganisms that secrete recombinant wild-type anaphylactic allergens, which could elicit a potentially lethal anaphylactic reaction in an individual.
D. Secretion Signals
In other embodiments of the present invention, expressed recombinant allergens (and/or immunomodulatory molecules, such as cytokines; see below) are secreted by the microorganisms. Preferably, secretion of the allergens occurs inside a mammalian cell to reduce or eliminate exposure of recombinant allergens to a subject's immune system. Secretion of recombinant allergens includes secretion into the extracellular medium and secretion into the periplasm of microorganisms such as gram-negative bacteria and yeast. Advantages of secreting recombinant allergens into the periplasm include reducing leakage of the allergens prior to phagocytosis of the microorganism. This advantage is most applicable in non-inducible systems. Advantages of secreting allergens into the extracellular medium in inducible systems include maximizing the amount of allergens available for processing by APCs after phagocytosis.
To express secreted recombinant allergens in bacteria, a variety of bacterial secretion signals known in the art may be used. For example, the Sec-dependent process in E. coli is one which is well known (for a review see Driessen et al. Curr. Opin. Microbiology. 1:216-22, 1998). In addition, the OmpA signal peptide in E. coli has been described by Wong and Sutherland (see U.S. Pat. No. 5,223,407). Fusion proteins containing either of these secretion signal peptides are not fully secreted by the bacteria, but rather transported across the inner membrane of the gram-negative bacteria into the periplasm. These secretion signals may be used in the present invention to transport recombinant allergens into the periplasm of bacteria. After administration of the inventive microorganisms to an individual and subsequent phagocytosis by APCs, the recombinant allergens in the periplasm are released after degradation of the outer membrane by enzymes in the endosome of the APCs. Preferably, the bacteria synthesize and secrete the polypeptides into the periplasm and are killed, preferably heat-killed, before administration. However, it is recognized that attenuated bacteria may also be used.
In another preferred embodiment, fusion proteins containing secretion signal sequences and recombinant allergen sequences are fully secreted into the extracellular medium by the microorganism after synthesis. Such secretion signals include those found in hemolysin and listeriolysin. In a particularly preferred embodiment, the hemolysin complex of E. coli is used to transport recombinant allergens across the inner and outer membrane of a microorganism (e.g., E. coli, Salmonella, Shigella, Vibrio, Yersinia, Citrobacter, Serratia, Pseudomonas) into the extracellular medium (Spreng et al. Mol. Microbiol. 31:1589-1601, 1999, and references therein all of which are incorporated herein by reference). Fusion of HlyAs to proteins has been shown to result in secretion of these fusion proteins utilizing the hemolysin secretion system (Blight and Holland, Trends Biotechnol. 12(11):450-5, 1994; Gentschev et al., Behring Inst Mitt. 95:57-66, 1994).
The hemolysin protein (HlyA) contains a C-terminal transport signal (HlyAs) which is approximately 50-60 amino acids in length (Hess et al., Mol Gen Genet. 224(2):201-8, 1990; Jarchau et al., Mol Gen Genet. 245(1):53-60, 1994). The HlyA protein is secreted across the inner and outer cellular membranes by the hemolysin secretion system. This complex contains three membrane proteins. Two of these proteins, HlyB and HlyD, are located in the inner membrane, and the third TolC, is located at the outer membrane. Genes encoding these proteins are part of the hemolysin operon which consists of four genes hlyC, hlyA, hlyB, and hlyD (Wagner et al., J Bacteriol. 154(1):200-10, 1983; Gentschev, Gene. 179(1):133-40, 1996). Ina preferred embodiment for use of the Hly secretion system, DNA plasmids (vectors) are used to express fusion proteins containing the HlyAs signal peptide and the recombinant allergen. The genes encoding the transport complex (hlyB, and hlyD) are encoded by the same vector. It is recognized that multiple vectors can be used to encode and express these genes, or that sequences encoding these genes can be inserted into the host genome for expression. Preferably, a single vector contains the complete hemolysin operon including the hly specific promoter and an enhancer-type regulator hlyR; the HlyA gene where only the minimal polypeptide sequence necessary to transport a fusion protein is present; and the recombinant allergen of interest. TolC protein is generally produced by the host E. coli system. However, in systems where tolC DNA is not encoded by a host organism, tolC can be encoded by a vector.
In a particularly preferred embodiment, the secretion plasmid pMOhly1 described in WO 98/50067 (“Donner”) is used to express fusion proteins containing secretion signal sequences and recombinant allergens of the invention. The secretion vector pMOhly1 contains the complete hemolysin operon including the hly specific promoter and an enhancer-type regulator hlyR. A majority of the hlyA gene has been deleted so that HlyA encodes only the 34 amino terminal and 61 carboxyl terminal amino acids (HlyAs). A unique Nsi restriction enzyme site between the amino terminal and carboxyl terminal residues of HlyA facilitates the insertion of heterologous genes or gene fragments into the reading frame of HlyAs. The genetic information for recombinant allergens of 10-1000 amino acids can be inserted into this secretion vector pMOhly1, which facilitates secretion in attenuated Salmonella and other gram-negative attenuated inoculation strains (e.g. E. coli, Vibrio cholera, Yersina enterocolitica). The secretion of fusion proteins using a single plasmid is described by Donner. An advantage of the hemolysin secretion system in comparison to conventional transport systems is the larger size of the fusion proteins that can be synthesized and secreted. Conventional secretion systems for the presentation of antigens are only capable of secreting relatively short peptides to the outer part of the bacterial cell (e.g., Cardenas and Clements, Clin Microbiol Rev. 5(3):328-42, 1992).
In certain preferred embodiments, microorganisms that secrete recombinant allergens are provided in association with an encapsulation device as described below in the context of the pharmaceutical compositions of the invention. Encapsulating the microorganisms in this manner provides an additional level of control over accidental exposure of recombinant allergens, particularly wild-type anaphylactic allergens, with IgE molecules bound to the surface of histamine-releasing mast cells and basophils. This further reduces or eliminates the risk of anaphylaxis during administration of microorganisms that produce anaphylactic allergens.
E. Pharmaceutical Compositions
As discussed above, the present invention provides microorganisms expressing recombinant versions of protein allergens that are useful for treating allergies and in particular anaphylactic allergies. Accordingly, in another aspect of the present invention, pharmaceutical compositions are provided, wherein these compositions comprise these microorganisms and a pharmaceutically acceptable carrier. Optionally the compositions include adjuvants and/or immunomodulatory molecules as discussed below. It will be appreciated that certain of the microorganisms of present invention may also be provided by combination or association with one or more other agents such as targeting agents or may be encapsulated as discussed in more detail below.
It will often be desirable to include microorganisms expressing recombinant forms of more than one protein allergen in a composition of the present invention. To give but one example, at least three different protein allergens, Ara h 1, Ara h 2 and Ara h 3, are thought to contribute to peanut allergy; >90% of individuals who are allergic to peanuts have IgE reactive with Ara h 1, >90% of allergic individuals have IgE reactive with Ara h 2 and >44% have IgE reactive with Ara h 3. As described in the Examples, inventive compositions may include a mixture of microorganisms that express recombinant forms of more than one of these proteins, or all of them. Also, it may be desirable to include recombinant forms of a variety of different kinds of protein allergens so that multiple allergies are treated simultaneously (e.g., without limitation, milk and peanut allergens, egg and peanut allergens, milk and egg allergens, etc.).
Pharmaceutically Acceptable Carriers
As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences Ed. by Gennaro, Mack Publishing, Easton, Pa., 1995, discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the microorganisms of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as hydroxypropyl cellulose, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator. Viscosity-enhancing carriers such as hydroxypropyl cellulose are preferred carriers of the invention for rectal administration (see discussion below) since they facilitate retention of the pharmaceutical composition within the rectum. In addition, in embodiments that involve rectal administration the volume of carrier that is added to the pharmaceutical composition is selected in order to maximize retention of the composition. In particular, the volume should not be so large as to jeopardize retention of the administered composition in the rectal vault.
Immunomodulatory Adjuvants or Molecules
In certain preferred embodiments of the invention, the microorganisms are provided in conjunction with one or more immunomodulatory adjuvants or molecules.
Those of ordinary skill in the art will readily appreciate preferred types of adjuvants for use with the inventive compositions. Preferred adjuvants are characterized by an ability to stimulate a Th1-type response preferentially over Th2-type response and/or to down regulate a Th2-type response. In particular, adjuvants that are known to stimulate Th2-type responses are avoided. In general, suitable adjuvants include gel-type adjuvants (e.g., aluminum hydroxide/aluminum phosphate, calcium phosphate), microbial adjuvants (e.g., immunomodulatory DNA sequences that include CpG motifs; endotoxins such as monophosphoryl lipid A; exotoxins such as cholera toxin, E. coli heat labile toxin, and pertussis toxin; and muramyl dipeptide); oil-emulsion and emulsifier-based adjuvants (e.g., Freund's Incomplete Adjuvant, MF59, and SAF); particulate adjuvants (e.g., liposomes, biodegradable microspheres, and saponins); and synthetic adjuvants (e.g., nonionic block copolymers, muramyl peptide analogues, polyphosphazene, and synthetic polynucleotides).
Immunomodulatory DNA sequences are adjuvants of particular interest (see, for example, U.S. Pat. No. 5,830,877; and WO96/02555, WO98/18810, WO98/16247 and WO98/40100). These immunomodulatory sequences of bacterial, viral, or invertebrate origin contain unmethylated CpG motifs and when injected into animals in conjunction with an antigen such as an allergen, appear to skew the immune response towards a Th1-type response. See, for example, Yamamoto et al., Microbiol. Immunol. 36:983, 1992; Krieg et al., Nature 374:546, 1995; Pisetsky, Immunity 5:303, 1996; and Zimmerman et al., J. Immunol. 160:3627, 1998. See also WO 00/54803, the contents of which are incorporated herein by reference. Other preferred adjuvants reported to induce Th1-type responses and not Th2-type responses include, for example, AVRIDINETM (N,N-dioctadecyl-N′N′-bis(2-hydroxyethyl)propanediamine) available from M6 Pharmaceuticals of New York, N.Y.; niosomes (non-ionic surfactant vesicles) available from Proteus Molecular Design of Macclesfield, UK; and CRL 1005 (a synthetic ABA non-ionic block copolymer) available from Vaxcel Corporation of Norcross, Ga. Particularly preferred adjuvants are ones that induce IL-12 production, including microbial extracts such as fixed Staphylococcus aureus, Streptococcal preparations, Mycobacterium tuberculosis, lipopolysaccharide (LPS), monophosphoryl lipid A (MPLA) from gram negative bacterial lipopolysaccharides (Richards et al. Infect Immun. 66(6):2859-65, 1998), Listeria monocytogenes, Toxoplasma gondii, and Leishmania major. Some polymers are also adjuvants. For example, polyphosphazenes are described in U.S. Pat. No. 5,500,161. These polymers can be used not only to encapsulate the microorganisms as described below but also to enhance the immune response to the recombinant allergen.
In general, immunomodulatory molecules include cytokines which are small proteins or biological factors (in the range of 5-20 kD) that are released by cells and have specific effects on cell-cell interaction, communication and behavior of other cells. Preferably, the cytokine(s) to be administered is/are selected to reduce production of a Th2 response. One preferred method of reducing a Th2 response is through induction of the alternative response. Cytokines that induce a Th1 response in T-cells include IL-1β, IL-2, IL-12, IL-18, IFN-α, IFN-γ and TNF-β.
In certain embodiments the immunomodulatory adjuvants and/or molecules are comprised or synthesized by the microorganisms of the invention. In other embodiments they may be provided as impure preparations (e.g., isolates of cells expressing a cytokine gene, either endogenous or exogenous to the cell) or purified preparations and mixed with the microorganisms. It is recognized that in preferred embodiments the microorganisms that are utilized to synthesize and deliver the recombinant allergens according to the present invention can act as adjuvants themselves.
Targeting Agents
Inventive compositions of the invention may desirably be associated with a targeting agent that will promote delivery to a particular desired location. In preferred embodiments of the invention, the microorganisms are targeted for uptake by APCs. For example, the microorganisms could be targeted to dendritic cells or macrophages via association with a ligand that interacts with an uptake receptor such as the mannose receptor or an Fc receptor. The microorganisms could be targeted to other APCs via association with a ligand that interacts with the complement receptor.
Alternatively or additionally, a microorganism could be targeted to particular vesicles within APCs. Those of ordinary skill in the art will appreciate that any targeting strategy should allow for proper uptake and processing of the microorganisms by the APCs.
A recombinant allergen of the present invention can be targeted by association of the microorganism with an Ig molecule, or portion thereof. Ig molecules are comprised of four polypeptide chains, two identical “heavy” chains and two identical “light” chains. Each chain contains an amino-terminal variable region and a carboxy-terminal constant region. The four variable regions together comprise the “variable domain” of the antibody; the constant regions comprise the “constant domain”. The chains associate with one another in a Y-structure in which each short Y arm is formed by interaction of an entire light chain with the variable region and part of the constant region of one heavy chain and the Y stem is formed by interaction of the two heavy chain constant regions with one another. The heavy chain constant regions determine the class of the antibody molecule and mediate the molecule's interactions with class-specific receptors on certain target cells; the variable regions determine the molecule's specificity and affinity for a particular antigen.
Class-specific antibody receptors, with which the heavy chain constant regions interact, are found on a variety of different cell types and are particularly concentrated on professional antigen presenting cells (pAPCs), including dendritic cells. According to the present invention, inventive compositions may be targeted for delivery to pAPCs through association with an Ig constant domain. In one embodiment, an Ig molecule is isolated whose variable domain displays specific affinity for a protein expressed on the surface of the microorganism to be delivered and the microorganism is delivered in association with the Ig molecule. The Ig may be of any class for which there is an Ig receptor, but in certain preferred embodiments, is an IgG. Also, it is not required that the entire Ig be utilized; any piece including a sufficient portion of the Ig heavy chain constant domain is sufficient. Thus, Fc fragments and single-chain antibodies may be employed in the practice of the present invention.
In one embodiment of the invention, a protein expressed on the surface of the microorganism is prepared as a fusion molecule with at least an Ig heavy chain constant region (e.g., with an Fc fragment), so that a single fusion protein, containing both the surface protein and Ig heavy chain constant region components, is exposed on the surface of the microorganism. This embodiment allows increased flexibility because the length and character of the surface protein is not constrained by the binding requirements of the Ig variable domain cleft. Fc fragments may be prepared by any available technique including, for example, recombinant expression (which may include expression of a fusion protein) proteolytic or chemical cleavage of Ig molecules (e.g., with papain), chemical synthesis, etc.
Encapsulation
In one particularly preferred embodiment of the invention, the inventive microorganisms are provided in association with an encapsulation device (see, for example, the encapsulation devices described in U.S. Patent Publication No. 2001-0031262 A1, incorporated herein by reference). Preferred encapsulation devices are biocompatible and stable inside the body so that the microorganisms and recombinant allergens are not released until after the encapsulation device is taken up into an APC. For example, preferred systems of encapsulation are stable at physiological pH and degrade at acidic pH levels comparable to those found in the endosomes of APCs. Preferably, the encapsulation device is taken up into APC via endocytosis in clathrin-coated pits. Particularly preferred encapsulation compositions include but are not limited to ones comprised of liposomes, polylactide-co-glycolide (PLGA), chitosan, synthetic biodegradable polymers, environmentally responsive hydrogels and/or gelatin PLGA nanoparticles. Inventive microorganisms may be encapsulated in combination with one or more immunomodulatory adjuvants or molecules and targeting entities. Alternatively or additionally the encapsulation device itself may be associated with a targeting agent and/or an immunomodulatory adjuvant or molecule.
Assays for Screening Inventive Compositions
In certain embodiments, once an inventive pharmaceutical composition has been prepared it may be assayed for its allergenicity. Both in vitro and in vivo assays for assessing the allergenicity of compositions are known to those skilled in the art. Conventional in vitro assays include RAST (Sampson and Albergo, J. Allergy Clin. Immunol. 74:26, 1984), ELISAs (Burks et al., N. Engl. J. Med. 314:560, 1986), immunoblotting (Burks et al., J. Allergy Clin. Immunol. 81:1135, 1988), basophil histamine release assays (Nielsen, Dan. Med. Bull. 42:455, 1995 and du Buske, Allergy Proc. 14:243, 1993) and others (Hoffmann et al., Allergy 54:446, 1999). Additionally or alternatively, the allergenicity of a composition may be assessed using an in vivo skin test (Sampson and Albergo, J. Allergy Clin. Immunol. 74:26, 1984). In certain preferred embodiments, the allergenicity of an inventive composition is assessed in vivo using a suitable animal model, without limitation a sensitized mouse. The preparation and use of animal models of allergies are described in WO 00/51647 and further in the Examples. When using an animal model to assess the allergenicity of an inventive composition, objective in vivo clinical symptoms may be monitored before and after the administration to determine any change in the clinical symptoms, changes in body temperature, changes in peak expiratory flow, etc. This is described in detail in Example 10. Preferably, an inventive composition exhibits minimal or no allergenicity under these tests (e.g., as compared to control and described in Example 10). In certain embodiments, if a composition is found to exhibit unfavorable levels of allergenicity it may prove advantageous to repeat the tests after washing the composition. According to such embodiments, low levels of exposed recombinant allergen may be responsible for the observed allergenicity and a simple washing step may be sufficient to remove these from the composition.
As noted previously, certain preferred compositions of the present invention are characterized by their ability to suppress a Th2-type response and/or to stimulate a Th1-type response preferentially as compared with their ability to stimulate a Th2-type response. Th1- and Th2-type responses are well-established alternative immune system responses that are characterized by the production of different collections of cytokines and/or cofactors that can be assayed for. For example, Th1-type responses are generally associated with production of cytokines such as IL-2, IL-6, IL-12, IL-18, IFN-α, IFN-γ and TNF-β by CD4+ T helper cells and the production of IgG antibodies. Exposure of CD4+ T-cells to allergens can also activate the cells to develop into Th2 cells, which secrete IL-4, IL-5, IL-10 and IL-13. The extent of T-cell subset suppression or stimulation may be determined by any available means including, for example, intra-cytoplasmic cytokine determination. In preferred embodiments of the invention, Th2 suppression is assayed, for example, by quantitation of IL-4, IL-5, IL-10 and/or IL-13 in stimulated T-cell culture supernatant or assessment of T-cell intra-cytoplasmic (e.g., by protein staining or analysis of mRNA) IL-4, IL-5, IL-10 and/or IL-13; Th1 stimulation is assayed, for example, by quantitation of IFN-α, IFN-γ, TNF-β, IL-2, IL-6, IL-12 and/or IL-18 in activated T-cell culture supernatant or assessment of intra-cytoplasmic levels of these cytokines Suitable cytokine assays are described in greater detail in Examples 12-13.
F. Uses of Pharmaceutical Compositions
In yet another aspect, according to the methods of treatment of the present invention, an individual who suffers from or is susceptible to an allergy may be treated with a pharmaceutical composition, as described herein. It will be appreciated that an individual can be considered susceptible to allergy without having suffered an allergic reaction to the particular protein allergen in question. For example, if the individual has suffered an allergic reaction to a related protein allergen (e.g., one from the same source or one for which shared allergies are common), that individual will be considered susceptible to allergic reaction to the relevant allergen. Similarly, if members of an individual's family react to a particular protein allergen, the individual may be considered to be susceptible to allergic reaction to that protein allergen. Individuals that are susceptible to an allergy but lack any relevant medical history can also be identified by a any known methods including: a prick skin test (Sampson and Albergo, J. Allergy Clin. Immunol. 74:26, 1984); measurement of serum titer of allergen-specific IgE (e.g., by RAST as described in Sampson and Albergo, J. Allergy Clin. Immunol. 74:26, 1984, by ELISA as described in Burks et al., N. Engl. J. Med. 314:560, 1986 or by immunoblotting as described in Burks et al., J. Allergy Clin. Immunol. 81:1135, 1988); basophil histamine release assays (Nielsen, Dan. Med. Bull. 42:455, 1995 and du Buske, Allergy Proc. 14:243, 1993) and other techniques (e.g., see Hoffmann et al., Allergy 54:446, 1999).
In general, it is believed that the inventive compositions will be clinically useful in treating or preventing allergic reactions associated with any protein allergen, in particular anaphylactic allergens including but not limited to food allergens, insect allergens and rubber allergens.
It will be appreciated that therapy or desensitization with the inventive compositions can be used in combination with any other known therapy for allergy, e.g., without limitation, allergen-non-specific anti-IgE antibodies that deplete the patient of allergen-specific IgE antibodies (see, Boulet et al., Am. J. Respir. Crit. Care Med. 155:1835, 1997; Fahy et al., Am. J. Respir. Crit. Care Med. 155:1828, 1997; and Demoly and Bousquet, Am J. Resp. Crit. Care Med. 155:1825, 1997).
It will further be appreciated that the therapeutic and prophylactic methods encompassed by the present invention are not limited to treating allergic reactions in humans, but may be used to treat allergies in any animal including but not limited to mammals, e.g., bovine, canine, feline, caprine, ovine, porcine, murine and equine species.
Therapeutically Effective Dose
The invention provides methods for the treatment or prevention of allergies comprising administering a therapeutically effective amount of an inventive pharmaceutical composition comprising a microorganism expressing a recombinant allergen to an individual in need thereof, in such amounts and for such time as is necessary to achieve the desired result. It will be appreciated that this encompasses administering an inventive pharmaceutical composition as a therapeutic measure to treat an individual who suffers from an allergy or as a prophylactic measure to desensitize an individual that is susceptible to an allergy. In this context, it has recently been demonstrated that pollen immunotherapy has a prophylactic effect in reducing the development of asthma in children with seasonal rhinoconjunticivitis (see Möller et al., J. Allergy Clin. Immunol. 109:251-6, 2002). In certain embodiments of the present invention a “therapeutically effective amount” of the pharmaceutical composition is that amount effective for preventing an allergic reaction in an individual who suffers from an allergy or an individual who is susceptible to an allergy. The pharmaceutical compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for preventing an allergic reaction. As described below, rectal and subcutaneous administration are preferred, rectal administration being particularly preferred. Thus, the expression “amount effective for preventing an allergic reaction”, as used herein, refers to a sufficient amount of pharmaceutical composition to prevent an allergic reaction. The exact dosage is chosen by the individual physician in view of the patient to be treated and the route of administration. Dosage and administration are adjusted to provide sufficient levels of the recombinant allergen or to maintain the desired effect. Additional factors which may be taken into account include the severity of the allergic reaction; age, weight and gender of the individual; diet, time and frequency of administration, therapeutic combinations, reaction sensitivities and tolerance/response to therapy. Treatment will typically be between twice a week and once a month, continuing for up to 3 months to 5 or more years, although this is highly dependent on the individual patient response. In general, therapeutically effective amounts will be in the microgram to milligram range of recombinant allergen.
In certain embodiments the dosage may be increased in steps, e.g., by doubling the dosage in a series of weekly administrations over an initial period (e.g., 4-16 weeks, preferably 6-10 weeks). As discussed in Example 14, an initial once weekly schedule of administration is a well-established immunotherapy paradigm for escalation to “maintenance” doses of immunotherapeutic extracts. In certain embodiments this may be followed with a biweekly or monthly schedule of administration at the final “high” dosage until the subject is desensitized (e.g., for 2-6 months or more, preferably 3-4 months). For example, without limitation, in certain embodiments, the compositions of the invention may be administered in increasing dosage levels until they reach about 0.1 μg to about 1,000 μg, preferably from about 1 μg to about 500 μg, more preferably 10 μg to about 100 μg of the recombinant allergen per kg of subject body weight. These dosage levels are extrapolated from those that have been shown to be safe and efficient in desensitizing peanut-allergic mice (see Examples 11-14). The increased spacing between administrations during the “maintenance” period may provide the immune system a sufficient period of time, with continued but not relentless exposure, to respond to the treatment and become desensitized. In certain embodiments it may prove advantageous to gradually decrease the dosage over time after this “maintenance” period until the patient is fully desensitized (e.g., as determined by a skin prick test, serum IgE levels, a supervised challenge with the natural allergen, etc.).
The recombinant allergens of the invention are preferably formulated in dosage unit form for ease of administration and uniformity of dosage. The expression “dosage unit form” as used herein refers to a physically discrete unit of recombinant allergen appropriate for the patient to be treated. It will be understood, however, that the total daily, weekly or monthly usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. For any inventive recombinant allergen, the therapeutically effective dose can be estimated initially either in cell culture assays or in non-human animal models, usually mice, rabbits, dogs, or pigs (e.g., see Examples 10-13). The non-human animal model is also used to achieve a desirable concentration range. Such information can then be used to determine useful doses for administration in humans (e.g., see discussion in Example 14 and above).
Administration of Pharmaceutical Compositions
After formulation with an appropriate pharmaceutically acceptable carrier in a desired dosage, the pharmaceutical compositions of this invention can be administered to humans and other mammals by a variety of routes. In particular the compositions can be administered topically (as by powders, ointments, or drops), orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, subcutaneously, intramuscularly, intragastrically, bucally, ocularly, or nasally, depending on the severity and nature of the allergic reaction being treated or prevented. Preferably the compositions are delivered parenterally, to the gastrointestinal tract (e.g., orally or rectally) or to mucosal tissues.
The inventors have established that subcutaneous and rectal delivery are particularly suitable delivery routes for the inventive compositions. As described in the Examples, administration of heat-killed E. coli cells expressing mutated Ara h 1, Ara h 2 and Ara h 3 peanut allergens was found to be more effective for the desensitization of peanut-allergic mice when the composition was administered subcutaneously or rectally. In addition, while some local inflammation was observed with subcutaneous delivery none was observed with rectal delivery. Thus rectal delivery is a particularly preferred route for administration.
Compositions for rectal administration are preferably suppositories which can be prepared by mixing the microorganisms of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectal vault and release the microorganisms (e.g., see Williams, Scand. J. Gastroenterol. Suppl. 172:60-2, 1990 and Torres-Lugo et al., Biomaterials 21(12):1191-6, 2000). Retention enemas and rectal catheters can also be used as is known in the art. Viscosity-enhancing carriers such as hydroxypropyl cellulose are also preferred carriers of the invention for rectal administration since they facilitate retention of the pharmaceutical composition within the rectum. Generally, the volume of carrier that is added to the pharmaceutical composition is selected in order to maximize retention of the composition. In particular, the volume should not be so large as to jeopardize retention of the administered composition in the rectal vault.
Injectable preparations (e.g., for subcutaneous administration) such as sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. Delayed absorption of a parenterally administered composition may be accomplished by dissolving or suspending the microorganisms in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the microorganisms in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of microorganisms to polymer and the nature of the particular polymer employed, the rate of microorganism release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the microorganisms in liposomes or microemulsions which are compatible with body tissues.
Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the microorganisms, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
Dosage forms for topical or transdermal administration of an inventive pharmaceutical composition include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, or patches. The microorganisms are admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. The ointments, pastes, creams and gels may contain, in addition to the microorganisms of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc, zinc oxide, or mixtures thereof.
Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the microorganisms are mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the microorganisms may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the microorganisms only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.
EXAMPLES
Allergy to peanuts is one of the most common and serious of the anaphylactic reactions to foods in terms of persistence and severity of reaction. Unlike the clinical symptoms of many other food allergies, the reactions to peanuts are rarely outgrown, therefore, most diagnosed children will have the disease for a lifetime (Sampson and Burks, Annu. Rev. Nutr. 16:161, 1996 and Bock, J. Pediatr. 107:676, 1985). The majority of cases of fatal food-induced anaphylaxis involve ingestion of peanuts (Sampson et al., NEJM 327:380, 1992 and Kaminogawa, Biosci. Biotech. Biochem. 60:1749, 1996). The only effective therapeutic option currently available for the prevention of a peanut hypersensitivity reaction is food avoidance. Unfortunately, for a ubiquitous food such as a peanut, the possibility of an inadvertent ingestion is great.
The major peanut allergen proteins Ara h 1, Ara h 2 and Ara h 3 were therefore chosen as gold-standards to illustrate various aspects of the present invention.
Ara h 1 has a molecular weight of about 63.5 kD and belongs to the vicilin family of seed storage proteins. The cloning and sequencing of Ara h 1 (Accession No. L34402 in GenBank) is described in Burks et al., J. Clin. Invest. 96:1715, 1995. The nucleotide sequence of a cDNA clone from that reference (cDNA clone P41b) is depicted in FIG. 1 (SEQ ID NO:1). The predicted amino acid sequence of the Ara h 1 protein encoded by cDNA clone P41b is depicted in FIG. 2 (SEQ ID NO:2).
Ara h 2 has a molecular weight of about 17 kD and belongs to the conglutin family of seed storage proteins. The cloning and sequencing of Ara h 2 (Accession No. L77197 in GenBank) is described in Stanley et al., Arch. Biochem. Biophys. 342:244, 1997. The nucleotide sequence of a cDNA clone from that reference (cDNA clone p38) is depicted in FIG. 3 (SEQ ID NO:3). The predicted amino acid sequence of the Ara h 2 protein encoded by cDNA clone p38 is depicted in FIG. 4 (SEQ ID NO:4).
Ara h 3 has a molecular weight of about 60 kD and belongs to the glycinin family of seed storage proteins. The cloning and sequencing of Ara h 3 (Accession No. AF093541 in GenBank) is described in Rabjohn et al., J. Clin. Invest. 103:535, 1999. The nucleotide sequence of a cDNA clone of Ara h 3 is depicted in FIG. 5 (SEQ ID NO:5). The predicted amino acid sequence of the protein encoded by this cDNA clone is depicted in FIG. 6 (SEQ ID NO:6).
Examples 1, 2 and 3 describe the mapping and mutational analysis of the linear IgE epitopes of Ara h 1, Ara h 2 and Ara h 3, respectively.
Example 4 describes the methods and constructs that were used to prepare and purify recombinant versions of Ara h 1, Ara h 2 and Ara h 3 (wild-type and mutant).
Example 5 describes in vitro experiments that were performed to compare the binding of wild-type Ara h 1 and mutant Ara h 1 with IgE sera from peanut-sensitive individuals.
Example 6 describes in vitro experiments that were performed to compare the binding of wild-type Ara h 2 and mutant Ara h 2 with IgE sera from peanut-sensitive individuals.
Example 7 describes in vitro cell-based mediator release experiments that were performed to compare the allergenicity of wild-type Ara h 2, mutant Ara h 2, native Ara h 2 purified from crude peanut extract, crude peanut extract, crude soybean extract and crude pea extract.
Example 8 describes in vitro experiments that were performed to compare the binding of wild-type Ara h 3 and mutant Ara h 3 with IgE sera from peanut-sensitive individuals.
Example 9 describes early in vitro and in vivo experiments that were performed to test the encapsulation of wild-type Ara h 1-3 expressed in E. coli. 
Example 10 describes in vivo safety experiments that were performed with sensitized mice to compare their reactions when challenged with CPE, HKE-P123, HKE-MP123, P123, MP123, and NP12.
Example 11 describes in vivo desensitization experiments that were performed with sensitized mice to compare the efficacy of different desensitizing protocols (i.e., different desensitizing compositions and delivery routes). The desensitization protocols that were compared included HKE-MP123 delivered subcutaneously, HKE-MP123 delivered intragastrically, HKE-MP123 delivered rectally, MP123 delivered rectally, HKL delivered subcutaneously, and HKL-MP123 delivered subcutaneously.
Example 12 describes in vivo desensitization experiments that were performed with sensitized mice to compare the efficacy of rectally delivered HKE-MP123 and MP123.
Example 13 describes in vivo desensitization experiments that were performed with sensitized mice to assess the long-term efficacy of rectally delivered HKE-MP123.
Example 14 describes a prophetic clinical study to demonstrate the safety and efficacy of rectally delivered HKE-MP123 in the treatment of human peanut-allergic patients.
Example 1
Mapping and Mutational Analysis of the Linear IgE Epitopes of Ara h 1
1.1 Introduction
Serum IgE from patients with documented peanut hypersensitivity reactions and overlapping peptides were used to identify the IgE binding epitopes on the major peanut allergen, Ara h 1. At least twenty-three different linear IgE binding epitopes, located throughout the length of the Ara h 1 protein, were identified. All of the epitopes were 6-10 amino acids in length, but there was no obvious sequence motif shared by all peptides. Four of the peptides appeared to be immunodominant IgE binding epitopes in that they were recognized by serum from more than 80% of the patients tested and bound more IgE than any of the other Ara h 1 epitopes. Mutational analysis of the epitopes revealed that single amino acid changes had dramatic effects on IgE binding characteristics.
1.2 Materials and Methods
Serum IgE
Serum from 15 patients with documented peanut hypersensitivity reactions (mean age=25 years) was used to identify the Ara h 1 IgE binding epitopes. Each of these individuals had a positive immediate prick skin test to peanut and either a positive double-blind placebo-controlled food challenge or a convincing history of peanut anaphylaxis (laryngeal edema, severe wheezing, and/or hypotension). Representative individuals with elevated serum IgE levels (who did not have peanut-specific IgE or peanut hypersensitivity) were used as controls in these studies. In some instances, a serum pool was made by mixing equal aliquots of serum IgE from each of the 15 patients with peanut hypersensitivity. This pool was then used in immunoblot analysis experiments to determine the IgE binding characteristics of the population. At least 5 ml venous blood was drawn from each patient and allowed to clot, and the serum collected. All studies were approved by the Human Use Advisory Committee at the University of Arkansas for Medical Sciences.
Computer Analysis of Ara h 1 Sequence
Analysis of the Ara h 1 amino acid sequence (clone P41b, SEQ ID NO:2) and peptide sequences was performed on the University of Arkansas for Medical Sciences' Vax computer using the Wisconsin DNA analysis software package. The predicted antigenic regions on the Ara h 1 protein are based on algorithms developed by Jameson and Wolf (Comput. Appl. Biosci. 4:181-186, 1988) that relate antigenicity to hydrophilicity, secondary structure, flexibility, and surface probability.
Peptide Synthesis
Individual peptides were synthesized on a derivatised cellulose membrane using Fmoc amino acid active esters according to the manufacturer's instructions (Genosys Biotechnologies, Woodlands, Tex.). Fmoc-amino acid derivatives were dissolved in 1-methyl-2-pyrrolidone and loaded on marked spots on the membrane. Coupling reactions were followed by acetylation with a solution of 4% (v/v) acetic anhydride in N,N-dimethylformamide (DMF). After acetylation, Fmoc groups were removed by incubation of the membrane in 20% (v/v) piperdine in DMF. The membrane was then stained with bromophenol blue to identify the location of the free amino groups. Cycles of coupling, blocking, and deprotection were repeated until the peptides of the desired length were synthesized. After addition of the last amino acid in the peptide, the amino acid side chains were deprotected using a solution of dichloromethane/trifluoroacetic acid/triisobutlysilane (1/1/0.05). Membranes were either probed immediately or stored at −20° C. until needed.
IgE Binding Assay
Cellulose membranes containing synthesized peptides were incubated with the serum pool or individual serum from patients with peanut hypersensitivity diluted (1:5) in a solution containing Tris/NaCl (10 mM Tris/HCl, 500 mM NaCl, pH 7.5) and 1% bovine serum albumin for at least 12 hours at 4° C. or 2 hours at room temperature. The primary antibody was detected with 125I-labeled anti-IgE antibody (Sanofi Pasteur Diagnostics, Chaska, Minn.).
1.3 Results
Identification of Multiple IgE Binding Epitopes within Ara h 1
The Ara h 1 amino acid sequence (SEQ ID NO:2) was first analyzed for potential antigenic epitopes using computer-based algorithms. There were 11 possible antigenic regions, each containing multiple antigenic sites, predicted by this analysis along the entire length of the molecule.
Preliminary experiments were then performed to map the major IgE binding regions of Ara h 1. Exo III digestion from the 5′ or 3′ end of a full length Ara h 1 cDNA clone was used to produce shortened clones whose protein products could then be tested for IgE binding by immunoblot analysis. All constructs bound IgE until they were reduced to the extreme carboxyl terminal (5′ Exo III) or amino terminal (3′ Exo III) end of the molecule. These results indicate that there are multiple IgE epitopes on the Ara h 1 allergen.
Seventy-seven overlapping peptides representing the entire length of the Ara h 1 protein were then synthesized to characterize the IgE binding regions in greater detail. Each peptide was fifteen amino acids long and offset from the previous peptide by eight amino acids. In this manner, the entire length of the Ara h 1 protein could be studied in large overlapping fragments. These peptides were then probed with a pool of serum IgE from 15 patients with documented peanut hypersensitivity or with serum IgE from a representative control patient with no food allergy. Serum IgE from the control patients did not recognize any of the synthesized peptides. In contrast, twelve IgE binding regions (D1-D12) along the entire length of the Ara h 1 protein were recognized by pooled IgE from the population of patients with peanut hypersensitivity. These IgE binding regions represent amino acid residues 35-72, 89-112, 121-176, 289-326, 337-350, 361-374, 393-416, 457-471, 489-513, 521-535, 544-583, and 593-607 of SEQ ID NO:2. In general, the computer predicted antigenic regions contained or were part of those that were determined by actual IgE binding. However, there were two predicted antigenic regions (between amino acids 221-230 and 263-278 of SEQ ID NO:2) that were not recognized by serum IgE from peanut hypersensitive individuals. In addition, there were numerous IgE binding regions found in the Ara h 1 protein between amino acids 450-600 of SEQ ID NO:2.
To determine the amino acid sequence of the IgE binding epitopes, small overlapping peptides spanning each of the larger IgE binding regions identified were synthesized. By synthesizing smaller peptides (ten amino acids long) that were offset from each other by only two amino acids, it was possible to identify individual IgE binding epitopes within the larger IgE binding regions of the Ara h 1 molecule. Table 1 summarizes the twenty-three IgE binding epitopes (SEQ ID NOs:7-29) and their respective positions within the Ara h 1 protein (SEQ ID NO:2).
The most common amino acids found were acidic (D, E) and basic (K, R) residues comprising 40% of all amino acids found in the IgE epitopes. There were no obvious amino acid sequence motifs shared by all the IgE epitopes.







TABLE 1







Ara h 1 IgE binding epitopes












SEQ ID

Amino acid
Ara h 1



NO:
Peptide
sequence1
positions2
















7
1
AKSSPYQKKT
25-34



 



8
2
QEPDDLKQKA
48-57



 



9
3
LEYDPRLVYD
65-74



 



10
4
GERTRGRQPG
89-98



 



11
5
PGDYDDDRRQ
 97-106



 



12
6
PRREEGGRWG
107-116



 



13
7
REREEDWRQP
123-132



 



14
8
EDWRRPSHQQ
134-143



 



15
9
QPRKIRPEGR
143-152



 



16
10
TPGQFEDFFP
294-303



 



17
11
SYLQEFSRNT
311-320



 



18
12
FNAEFNEIRR
325-334



 



19
13
EQEERGORRW
344-353



 



20
14
DITNPINLRE
393-402



 



21
15
NNFGKLFEVK
409-418



 



22
16
GTGNLELVAV
461-470



 



23
17
RRYTARLKEG
498-507



 



24
18
ELHLLGFGIN
525-534



 



25
19
HRIFLAGDKD
539-548



 



26
20
IDQIEKQAKD
551-560



 



27
21
KDLAFPGSGE
559-568



 



28
22
KESHFVSARP
578-587



 



29
23
PEKESPEKED
597-606







1The underlined portions of each peptide represent the linear IgE epitopes.



2The Ara h 1 amino acid positions are taken from SEQ ID NO: 2.






Identification of Immunodominant Ara h 1 Epitopes

In an effort to determine which, if any, of the twenty-three epitopes was immunodominant, each set of twenty-three peptides was probed individually with serum IgE from ten different patients. Serum from five individuals randomly selected from the fifteen patient serum pool and an additional five sera from peanut-hypersensitive patients not represented in the serum pool were used to identify the commonly recognized epitopes. Immunoblot strips containing peptides 1-23 (see Table 1) were incubated with each individual patient's serum. The intensity of IgE binding to each spot was determined as a function of that patient's total IgE binding to these twenty-three epitopes. All of the patient sera tested (10/10) recognized multiple peptides. The most commonly recognized peptides were those that contained epitopes 1, 3, 4, 13, 17 and 22. These epitopes were recognized by IgE from at least 80% of the patient sera tested (8/10). In addition, epitopes 1-4, 8, 12, and 17, when recognized, bound more serum IgE from individual patients than any of the other epitopes. These results suggest that peptides 1, 3, 4, and 17 contain the immunodominant epitopes of the Ara h 1 protein.
Mutational Analysis of Ara h 1 Epitopes
The specific amino acids involved in IgE binding were determined by synthesizing duplicate peptides with single amino acid changes at each position. These peptides were then probed with pooled serum IgE from fifteen patients with peanut hypersensitivity to determine if the changes affected peanut-specific IgE binding. In general, each epitope could be mutated to a non-IgE binding peptide by the substitution of an alanine or methionine for a single amino acid residue. There was no obvious position within each peptide that, when mutated, would result in loss of IgE binding. Furthermore, there was no consensus in the type of amino acid that, when changed to alanine or methionine, would lead to loss of IgE binding. Table 2 summarizes these results.
The amino acids within each epitope were classified according to whether they were hydrophobic, polar, or charged residues. There were a total of 196 amino acids present in the twenty-one epitopes of Ara h 1 that were studied (epitopes 16 and 23 were not included in this study because they were recognized by a single patient who was no longer available to the study). Charged residues occurred most frequently (89/196), with hydrophobic residues (71/196) being the next frequent type of amino acid in the epitopes, and polar residues representing the least frequent amino acid group (36/196). 35% of the mutated hydrophobic residues resulted in loss of IgE binding (<1% IgE binding), whereas only 25 and 17% of mutated polar and charged residues, respectively, had a similar effect. These results indicated that the hydrophobic amino acid residues within these IgE binding epitopes were the most sensitive to changes. In addition results from this analysis indicated that the amino acids located near the center of the epitope were more critical for IgE binding.







TABLE 2







Amino acids mutations that reduce IgE binding


to Ara h 1












SEQ ID

Amino acid
Ara h 1



NO:
Peptide
sequence1
positions2
















7
1
AKSSPYQKKT
25-34



 



8
2
QEPDDLKQKA
48-57



 



9
3
LEYDPRLVYD
65-74



 



10
4
GERTRGRQPG
89-98



 



11
5
PGDYDDDRRQ
 97-106



 



12
6
PRREEGGRWG
107-116



 



13
7
REREEDWRQP
123-132



 



14
8
EDWRRPSHQQ
134-143



 



15
9
QPRKIRPEGR
143-152



 



16
10
TPGQFEDFFP
294-303



 



17
11
SYLQEFSRNT
311-320



 



18
12
FNAEFNEIRR
325-334



 



19
13
EQEERGQRRW
344-353



 



20
14
DITNPINLRE
393-402



 



21
15
NNFGKLFEVK
409-418



 



23
17
RRYTARLKEG
498-507



 



24
18
ELHLLGFGIN
525-534



 



25
19
HRIFLAGDKD
539-548



 



26
20
IDQIEKQAKD
551-560



 



27
21
KDLAFPGSGE
559-568



 



28
22
KESHFVSARP
578-587







1The amino acids that, when altered, lead to loss of IgE binding are shown as the bold, underlined residues. Epitopes 16 and 23 were not included in this study because they were recognized by a single patient who was no longer available to the study.



2The Ara h 1 amino acid positions are taken from SEQ ID NO: 2.






1.4 Conclusion
Multiple antigenic sites were predicted for the Ara h 1 allergen based on a computational analysis. At least twenty-three different linear IgE epitopes have been identified within the major peanut allergen Ara h 1. These sites are distributed throughout the protein.
Four of the Ara h 1 epitopes appear to be immunodominant IgE binding epitopes in that they are recognized by more than 80% of patient sera tested and, when recognized, bind more serum IgE from individual patients than any of the other epitopes. Epitope 17, which is located in the C-terminal end of the protein (amino acids 498-507 of SEQ ID NO:2), is in a region that shares significant sequence similarity with vicilins from other legumes (Gibbs et al., Mol. Biol. Evol. 6:614-623, 1989). The amino acids important for IgE binding also appear to be conserved in this region and may explain the possible cross-reacting antibodies to other legumes that can be found in sera of patients with a positive double-blind placebo-controlled food challenge to peanuts. Epitopes 1, 3, and 4 located in the N-terminal portion of the protein (amino acids 25-34, 65-74, and 89-98 of SEQ ID NO:2), appear to be unique to this peanut vicilin and do not share any significant sequence similarity with vicilins from other legumes (Gibbs et al., 1989, supra). The amino acids important to IgE binding in this region are not conserved. Hydrophobic amino acid residues appear to play the most important role in immunoglobulin binding.
Example 2
Mapping and Mutational Analysis of the Linear IgE Epitopes of Ara h 2
2.1 Introduction
The major linear IgE-binding epitopes of this allergen were mapped using overlapping peptides synthesized on an activated cellulose membrane and pooled serum IgE from fifteen peanut-sensitive patients. Ten IgE-binding epitopes were identified, distributed throughout the length of the Ara h 2 protein. 63% of the amino acids represented in the epitopes were either polar uncharged or apolar residues. In an effort to determine which, if any, of the ten epitopes were recognized by the majority of patients with peanut hypersensitivity, each set of ten peptides was probed individually with serum IgE from ten different patients. All of the patient sera tested recognized multiple epitopes. Three epitopes (epitopes 3, 6 and 7) were recognized by all patients tested. In addition, these three peptides bound more IgE than all the other epitopes combined, indicating that they are the immunodominant epitopes of the Ara h 2 protein. Mutational analysis of the Ara h 2 epitopes indicates that single amino acid changes result in loss of IgE binding. Two epitopes in the region spanning amino acids 57-74 of SEQ ID NO:4 contained the amino acid sequence DPYSPS (SEQ ID NO:30) that appears to be involved in IgE binding.
2.2 Materials and Methods
Serum IgE, Peptide Synthesis and IgE Binding Assay
Serum IgE was selected as described in Example 1, Section 1.2. Peptides were synthesized as described in Example 1, Section 1.2. The IgE binding assay was performed as described in Example 1, Section 1.2.
2.3 Results
Identification of Multiple IgE Binding Epitopes within Ara h 2
Nineteen overlapping peptides representing the derived amino acid sequence of the Ara h 2 protein were synthesized to determine which regions were recognized by serum IgE. Each peptide was fifteen amino acids long and was offset from the previous peptide by eight amino acids. In this manner, the entire length of the Ara h 2 protein could be studied in large overlapping fragments. These peptides were then probed with a pool of serum from fifteen patients with documented peanut hypersensitivity or serum from a representative control patient with no peanut hypersensitivity (see Example 1). Serum IgE from the control patient did not recognize any of the synthesized peptides. In contrast, three IgE binding regions within the Ara h 2 protein were recognized by the population of patients with peanut hypersensitivity. These IgE-binding regions represent amino acid residues 17-39, 41-80, and 114-157 of SEQ ID NO:4.
In order to determine the exact amino acid sequence of the IgE binding regions, smaller peptides (ten amino acids long offset by two amino acids) representing the larger IgE-binding regions were synthesized. In this manner it was possible to identify individual IgE-binding epitopes within the larger IgE-binding regions of the Ara h 2 molecule. The ten IgE-binding epitopes that were identified in this manner are shown in Table 3. The size of the epitopes ranged from 6 to 10 amino acids in length.







TABLE 3







Ara h 2 IgE binding epitopes












SEQ ID

Amino acid
Ara h 2



NO:
Peptide
sequence1
positions2
















31
1
HASARQQWEL
15-24



 



32
2
QWELQGDRRC
21-30



 



33
3
DRRCQSQLER
27-36



 



34
4
LRPCEQHLMQ
39-48



 



35
5
KIQRDEDSYE
49-58



 



36
6
YERDPYSPSQ
57-66



 



37
7
SQDPYSPSPY
65-74



 



38
8
DRLQGRQQEQ
115-124



 



39
9
KRELRNLPQQ
127-136



 



40
10
QRCDLDVESG
143-152







1The underlined portions of each peptide represent the linear IgE epitopes.



2The Ara h 2 amino acid positions are taken from SEQ ID NO: 4.






Three epitopes (epitopes 1-3), which partially overlapped with each other, were found in the region of amino acid residues 17-39 of SEQ ID NO:4. Four epitopes (epitopes 4-7) were found in the region represented by amino acid residues 41-80 of SEQ ID NO:4. Finally, three epitopes (epitopes 8-10) were found in the region represented by amino acid residues 114-157 of SEQ ID NO:4. 63% of the amino acids represented in the epitopes were either polar uncharged or apolar residues. There was no obvious amino acid sequence motif that was shared by all the epitopes, with the exception of epitopes 6 and 7, which contained the sequence DPYSPS (SEQ ID NO:30).
Identification of Immunodominant Ara h 2 Epitopes
In an effort to determine which, if any, of the ten epitopes was immunodominant, each set of ten peptides was probed individually with serum IgE from ten different patients. Five patients were randomly selected from the pool of fifteen patients used to identify the common epitopes, and five patients were selected from outside this pool. The intensity of IgE binding to each spot was determined as a function of that patient's total IgE binding to the ten epitopes. All of the patient sera tested (10/10) recognized multiple peptides. Peptides 3, 6 and 7 were recognized by serum IgE of all patients tested (10/10). In addition, serum IgE that recognized these peptides represented the majority of Ara h 2 specific IgE found in these patients. These results suggest that peptides 3, 6, and 7 contain immunodominant IgE epitopes of the Ara h 2 protein.
Mutational Analysis of Ara h 2 Epitopes
The specific amino acids involved in IgE binding were determined by synthesizing duplicate peptides with single amino acid changes at each position. These peptides were then probed with pooled serum IgE from fifteen patients with documented peanut hypersensitivity. In general, each peptide could be mutated to a non-IgE-binding peptide by the substitution of an alanine for a single amino acid residue. Table 4 summarizes these results. There was no obvious position within each peptide that, when mutated, would result in loss of IgE binding. Furthermore, there was no consensus in the type of amino acid that, when changed to alanine, would lead to loss of IgE binding.







TABLE 4







Amino acids mutations that reduce IgE binding


to Ara h 2












SEQ ID

Amino acid
Ara h 2



NO
Peptide
sequence1
positions2
















31
1
HASARQQWEL
15-24



 



32
2
QWELQGDRRC
21-30



 



33
3
DRRCQSQLER
27-36



 



34
4
LRPCEQHLMQ
39-48



 



35
5
KIQRDEDSYE
49-58



 



36
6
YERDPYSPSQ
57-66



 



37
7
SQDPYSPSPY
65-74



 



38
8
DRLQGRQQEQ
115-124



 



39
9
KRELRNLPQQ
127-136



 



40
10
QRCDLDVESG
143-152







1The amino acids that, when altered, lead to loss of IgE binding are shown as the bold, underlined residues.



2The Ara h 2 amino acid positions are taken from SEQ ID NO: 4.






2.4 Conclusion
There are at least ten IgE recognition sites distributed throughout the major peanut allergen Ara h 2. Two epitopes in Ara h 2 share a hexameric peptide (DPYSPS, SEQ ID NO:30). Both of these peptides are recognized by serum IgE from all the peanut hypersensitive patients tested in this study. In addition, serum IgE that recognize these peptides represent the majority of Ara h 2-specific IgE found in these patients.
Example 3
Mapping and Mutational Analysis of the Linear IgE Epitopes of Ara h 3
3.1 Introduction
Serum IgE from peanut allergic patients and overlapping, synthetic peptides were used to map the linear, IgE epitopes of Ara h 3. Several epitopes were found within the primary sequence, with no obvious sequence motif shared by the peptides. One epitope was recognized by all peanut-allergic patients. Mutational analysis of the epitopes revealed that single amino acid changes within these peptides could lead to a reduction or loss of IgE binding.
3.2 Materials and Methods
Serum IgE, Peptide Synthesis and IgE Binding Assay
Serum IgE was selected as described in Example 1, Section 1.2. Peptides were synthesized as described in Example 1, Section 1.2. The IgE binding assay was performed as described in Example 1, Section 1.2.
3.3 Results
Identification of Multiple IgE Binding Epitopes within Ara h 3
Sixty three overlapping peptides were synthesized to determine the regions of Ara h 3 that are recognized by serum IgE. Each peptide synthesized was fifteen amino acids long and offset from the previous peptide by eight amino acids. This approach allowed the analysis of the entire Ara h 3 primary sequence in large, overlapping fragments. These peptides were probed with a serum pool of IgE from peanut-hypersensitive patients who had previously been shown to recognize recombinant Ara h 3. Four IgE-binding regions were identified within the Ara h 3 primary amino acid sequence. These IgE-binding regions were represented by amino acid residues 21-55, 134-154, 231-269, and 271-328 of SEQ ID NO:6. To determine the exact amino acid sequence of the IgE-binding regions, synthetic peptides (fifteen amino acids offset by two amino acids) representing the larger IgE-binding regions were generated and probed with a serum pool of IgE from peanut allergic patients. This process made it possible to distinguish individual IgE-binding epitopes within the larger IgE-binding regions of the Ara h 3 protein. Four IgE-binding epitopes were identified in this manner and are shown in Table 5.







TABLE 5







Ara h 3 IgE binding epitopes











SEQ






ID
Pep-
Amino acid
Ara h 3



NO.
tide
sequence
positions1
Recognition2





41
1
IETWNPNNQEFECAG
33-47
 25% (2/8)


 


42
2
GNIFSGFTPEFLEQA
240-254
 38% (3/8)


 


43
3
VTVRGGLRILSPDRK
279-293
100% (8/8)


 


44
4
DEDEYEYDEEDRRRG
303-317
 38% (3/8)





1The Ara h 3 amino acid positions are taken from SEQ ID NO: 6.


2The percent recognition is the percentage of patients previously shown to recognize recombinant Ara h 3 whose serum IgE recognized that particular synthetic epitope.






Characterization of the IgE binding regions was repeated using synthetic overlapping peptides which were ten amino acids in length and offset by two amino acids. As with the 15/2 peptides, the 10/2 peptides were probed with a serum pool of IgE from patients who recognized recombinant Ara h 3. The four IgE-binding epitopes identified in this manner are shown in Table 6.







TABLE 6







Ara h 3 IgE binding epitopes











SEQ ID

Amino acid
Ara h 3



NO.
Peptide
sequence
positions1
Recognition2





45
5
EQEFLRYQQQ
183-192
  5% (1/20)


 


46
6
FTPEFLEQAF
246-255
 25% (5/20)


 


47
7
EYEYDEEDRR
306-315
 35% (7/20)


 


48
8
LYRNALFVAH
379-388
100% (20/20)





1The Ara h 3 amino acid positions are taken from SEQ ID NO: 6.


2The percent recognition is the percentage of patients previously shown to recognize recombinant Ara h 3 whose serum IgE recognized that particular synthetic epitope.






Identification of Immunodominant Ara h 3 Epitopes

To determine whether any of the four epitopes of Table 5 were immunodominant, each set of four peptides was probed individually with serum IgE from the eight patients previously shown to recognize recombinant Ara h 3 (results summarized in Table 5 as percentage recognition). Epitope 1 was recognized by serum IgE form 25% (2/8) of the patients tested, whereas epitopes 2 and 4 were recognized by serum IgE from 38% (3/8) of the eight patients tested. Epitopes 2 and 4 were recognized by the same three patients. Epitope 3 was recognized by serum IgE from 100% (8/8) of the peanut-allergic patients, classifying it as an immunodominant epitope within the peanut-allergic population. 68% of the amino acids constituting the epitopes were either polar uncharged or apolar residues. However, three was no obvious sequence motif with respect to position or polarity shared by the individual epitopes.
To determine whether any of the four epitopes of Table 6 were immunodominant, each set of four peptides was probed individually with serum IgE form a larger group of twenty patients previously shown to recognize recombinant Ara h 3 (results summarized in Table 6 as percentage recognition).
Mutational Analysis of Ara h 3 Epitopes
The specific amino acids involved in IgE binding to the Ara h 3 epitopes of Table 5 were determined by synthesizing multiple peptides with single amino acid changes at each position. These peptides were probed with a pool of serum IgE from patients who had previously recognized the wild-type peptide, to determine whether amino acid changes affected peanut-specific IgE binding. In general, each epitope could be altered to a non-IgE-binding peptide by the replacement of the wild-type amino acid residue with alanine The results are shown in Table 7.







TABLE 7







Amino acids mutations that reduce IgE binding


to Ara h 3












SEQ ID

Amino acid
Ara h 3



NO
Peptide
sequence1
position2







41
1
IETWNPNNQEFECAG
33-47



 



42
2
GNIFSGFTPEFLEQA
240-254



 



43
3
VTVRGGLRILSPDRK
279-293



 



44
4
DEDEYEYDEEDRRRG
303-317







1The amino acids that, when altered, lead to loss of IgE binding are shown as the bold, underlined residues.



2The Ara h 3 amino acid positions are taken from SEQ ID NO: 6.






It appears that the central amino acids within each epitope are favored for mutation. All mutations that led to a significant decrease in IgE binding were located at residues found within each core epitope. There was no obvious consensus in the type of amino acid that, when mutated to alanine, leads to complete loss or a decrease in IgE binding.
3.4 Conclusion
By generating synthetic, overlapping peptides representing the entire primary sequence of Ara h 3, we were able to determine that there are several IgE-recognition sites distributed throughout the primary sequence of the protein. One of these sites (within peptide 3 of Table 5) was recognized by serum IgE from every peanut-allergic patient in the group, designating it as an immunodominant epitope. Epitopes located within peptides 3 and 4 (Table 5) are located within the hypervariable region of the acidic chain, a stretch of amino acids that is highly variable in length among 11S storage proteins. This region contains a high proportion of glutamate, aspartate, and arginine residues and will tolerate large, naturally occurring insertions or deletions. Computer predictions from other studies suggest that this region is exposed on the surface of the protein (Nielsen et al., pp. 635-640 in “NATO Advanced Study Institute on Plant Molecular Biology”, Ed. by R. Hermann and B. Larkins, Plenum Press, New York, N.Y., 1990).
Example 4
Bacterial Expression and Purification of Recombinant Ara h 1-3
4.1 Introduction
This Example describes the constructs and methods that were used to express and purify the recombinant Ara h 1, Ara h 2 and Ara h 3 proteins that were used in the Examples that follow (both “wild-type” and “mutant”). Briefly, E. coli cells (BL21 or BLR) were transformed with Ara h 1, Ara h 2 or Ara h 3 constructs and exponentially growing cells were induced with IPTG (isopropyl-beta-D-thiogalactopyranoside). Cells were then pelleted and the recombinant proteins purified by affinity chromatography on a Ni2+-resin column.
4.2 Expression Vector Constructs for Ara h 1
“Wild-Type” Ara h 1
The portion of Ara h 1 sequence (SEQ ID NO:1) excluding the first 66 nucleotides, which encodes the signal peptide, was amplified by PCR. The PCR product was ligated into the cloning region of pET24b(+) (Novagen, Madison, Wis.) that carries a selectable marker for kanamycin resistance. The pET24b(+) vector also encodes a T7-tag (MASMTGGQQMG, SEQ ID NO:49) and a His-tag (HHHHHH, SEQ ID NO:50) that are added to the N- and C-termini, respectively, of the resulting recombinant protein. Some vector derived amino acids (RDPNSSS, SEQ ID NO:51) were included in between the T7-tag and the N-terminus of the Ara h 1 sequence. Some vector derived amino acids (KLAAALE, SEQ ID NO:52) were also included in between the His-tag and the C-terminus of the Ara h 1 sequence. The amino acid sequence of the recombinant Ara h 1 allergen is shown in FIG. 7 (SEQ ID NO:53). This “wild-type” recombinant Ara h 1 allergen included all the linear IgE epitopes that were identified in Example 1.
“Mutant” Ara h 1
Mutant recombinant Ara h 1 was prepared as above except that certain amino acids that were shown to be important for IgE binding in Example 1 were mutated by single-stranded mutagenesis and/or by PCR. Mutations were confirmed by sequence analysis of recombinant Ara h 1 cDNA clones. Three different mutants were prepared (MUT1, MUT2 and MUT3). MUT1 included single mutations in the four immunodominant epitopes (epitopes 1, 3, 4 and 17) and three non-immunodominant epitopes (epitopes 2, 5 and 6). Epitopes 1-6 were also chosen for mutation because they lie within the variable N-terminal domain and are not conserved between vicilins and therefore may be responsible for the extreme allergenicity to peanuts. MUT2 and MUT3 included mutations in twenty-one of the twenty-three linear IgE epitopes that were identified in Example 1 and further in a new epitope that was not identified in Example 1. This new epitope (shown with a *) spans amino acids 365-385 of SEQ ID NO:2. Epitopes 16 and 23 from Example 1 were not mutated since these were only recognized by a single allergic patient that was no longer available for study. MUT2 included some double mutations (epitopes 17 and 21), and a few accidental mutations outside the linear IgE epitopes. The mutations and their locations within the Ara h 1 sequence (SEQ ID NO:2) are listed in Table 8. MUTT was used in Example 6. MUT2 was used in all other Examples.







TABLE 8







Substitutions in mutant recombinant Ara h 1










1Epitope
3MUT1
2,3MUT2
3MUT3













1
K32A
K32A
K32A


2
D52A
D52A
D52A


3
V72A
V72A
V72A


4
R91A
R91A
R91A


5
D103A
D103A
D103A


6
R109A
R109A
R109A


7
—
W129A
W129A


8
—
R137A
R137A


9
—
I147A
I147A


—
—
W158C
—


—
—
T246I
—


—
—
E260D
—


10
—
F298M
F298M


11
—
S317A
S317A


12
—
F329A
F329A


13
—
E347A
E347A


*
—
K370D
—


*
—
V373S
V373S


14
—
I398A
I398A


15
—
F415A
F415A


316
—
—
—


—
—
Q475R
—


17
R499A
R499A
R499A


17
—
K505R
—


18
—
H527A
H527A


19
—
K547A
K547A


20
—
D560A
D560A


21
—
F563A
F563A


21
—
G567C
—


22
—
S584A
S584A


323
—
—
—





* This represents a new epitope that was not identified in Example 1 and that spans amino acids 365-385 of SEQ ID NO: 2.


1Epitopes identified in Example 1.


2MUT2 included a few accidental mutations outside the linear IgE epitopes of Ara h 1.


3Epitopes 16 and 23 were not mutated since they were only recognized by IgE from a single patient that was no longer available for study (see Example 1).


3The Ara h 1 amino acid positions are taken from SEQ ID NO: 2.






4.3 Expression Vector Constructs for Ara h 2
“Wild-Type” Ara h 2
The portion of Ara h 2 sequence (SEQ ID NO:3) excluding the first 54 nucleotides, which encodes part of the signal peptide, was amplified by PCR. The PCR product was ligated into the cloning region of pET24a(+) (Novagen, Madison, Wis.) that carries a selectable marker for kanamycin resistance. The pET24a(+) vector also encodes a T7-tag (MASMTGGQQMG, SEQ ID NO:49) and a His-tag (HHHHHH, SEQ ID NO:50) that are added to the N- and C-termini, respectively, of the resulting recombinant protein. Some vector derived amino acids (RGSEF, SEQ ID NO:54) were included in between the T7-tag and the N-terminus of the Ara h 2 sequence. Some vector derived amino acids (AAALE, SEQ ID NO:55) were also included in between the His-tag and the C-terminus of the Ara h 2 sequence. The amino acid sequence of the recombinant Ara h 2 allergen is shown in FIG. 8 (SEQ ID NO:56). This “wild-type” recombinant Ara h 2 allergen included all the linear IgE epitopes that were identified in Example 2.
“Mutant” Ara h 2
Mutant recombinant Ara h 2 was prepared as above except that certain amino acids that were shown to be important for IgE binding in Example 2 were mutated by single-stranded mutagenesis and/or by PCR. Mutations were confirmed by sequence analysis of recombinant Ara h 2 cDNA clones. Four different mutants MUT1, MUT2, MUT3 and MUT4 were prepared. MUT1 included single mutations in the three immunodominant epitopes (epitopes 3, 6 and 7) and one non-immunodominant epitope (epitope 4). MUT2 included single mutations in the three immunodominant epitopes (epitopes 3, 6 and 7) and two non-immunodominant epitopes (epitope 1 and 2 that overlap at position 23 of SEQ ID NO:4). MUT3 and MUT4 included mutations in all ten linear IgE epitopes that were identified in Example 2. MUT3 included three mutations within epitope 4. The mutations and their locations within the Ara h 2 sequence (SEQ ID NO:4) are listed in Table 9. MUT1, MUT2 and MUT3 were all used in Example 7. MUT3 only was used in all other Examples.







TABLE 9







Substitutions in mutant recombinant Ara h 2











1Epitope
2MUT1
2MUT2
2MUT3
2MUT4





1, 2
—
Q23A
—
—


1, 2
—
—
W25A
W25A


3
—
Q34A
—
—


3
E38A
—
E38A
E38A


4
P44A
—
P44A
P44A


4
—
—
E46A
—


4
—
—
Q51R
—


5
—
—
D56A
D56A


6
D63A
D63A
D63A
D63A


7
D70A
D70A
D70A
D70A


8
—
—
R123A
R123A


9
—
—
L133A
L133A


10 
—
—
L150A
L150A





1Epitopes identified in Example 2.


2The Ara h 2 amino acid positions are taken from SEQ ID NO: 4.






4.4 Expression Vector Constructs for Ara h 3
“Wild-Type” Ara h 3
The Ara h 3 sequence (SEQ ID NO:5) was amplified by PCR (the signal peptide is not encoded by this particular cDNA clone). The PCR product was ligated into the cloning region of pET24b(+) (Novagen, Madison, Wis.) that carries a selectable marker for kanamycin resistance. The pET24b(+) vector also encodes a T7-tag (MASMTGGQQMG, SEQ ID NO:49) and a His-tag (HHHHHH, SEQ ID NO:50) that are added to the N- and C-termini, respectively, of the resulting recombinant protein. Some vector derived amino acids (VDKLAAALE, SEQ ID NO:57) were included in between the His-tag and the C-terminus of the Ara h 3 sequence. The amino acid sequence of this “wild-type” recombinant Ara h 3 allergen is shown in FIG. 9 (SEQ ID NO:58).
“Mutant” Ara h 2
Mutant recombinant Ara h 3 was prepared as above except that certain amino acids that were shown to be important for IgE binding in Example 3 were mutated by single-stranded mutagenesis and/or by PCR. Mutations were confirmed by sequence analysis of recombinant Ara h 3 cDNA clones. Two different mutants MUT1 and MUT2 were prepared. MUT1 and MUT2 included mutations in all four linear IgE epitopes that were identified in Table 5 of Example 3. MUT1 included two mutations within epitope 4. The mutations and their locations within the Ara h 3 sequence (SEQ ID NO:6) are listed in Table 10. MUT1 was used in all of the following Examples.







TABLE 10







Substitutions in mutant recombinant Ara h 3









1Epitope
2-4MUT1
2MUT2





1
P58A
P58A


2
F270A
F270A


3
I307A
I307A


4
E328A
E328A


4
D333V
—





1Epitopes identified in Example 3.


2The Ara h 3 amino acid positions are taken from SEQ ID NO: 6.


3MUT1 only included the first three residues from the “MASMTGGQQMG” T7-tag.


4MUT1 lacked the first “I” amino acid of the Ara h 3 sequence.






4.5 Expression
Preparation for Induction
Transformed E. coli (BL21 or BLR) cells were picked from a glycerol freezer stock using a sterile toothpick and then inoculated in 100 ml LB broth (Luria-Bertani) containing 30 μg/ml of Kanamycin. The cells were then incubated overnight with shaking at 37° C. 20 ml of the incubated culture was then used to inoculate 1000 ml LB broth containing 30 μg/ml of Kanamycin.
Induction
The culture was then incubated with shaking at 37° C. until the O.D. at 600 nm reached 0.6. Typically this took about 2.5 hours. 1 ml of a 1 M stock solution of IPTG (isopropyl-beta-D-thiogalactopyranoside) was then added to the culture to give a final concentration of 1 mM and incubation was continued overnight. The cells were harvested by centrifugation at 5000 g for 15 minutes at 4° C. (5,5000 rpm in a Sorvall GSATM rotor). The harvested cells were passed onto purification or stored as frozen pellets at −70° C.
4.6 Purification
Cell Lysis
Harvested cells were re-suspended in 600 ml of 0.1% NONIDET® P40 in binding buffer (0.5 M NaCl, 20 mM Tris-HCl, and 6 M urea at pH 7.9) and then sonicated on ice for 20 minutes using a model 500 Sonic Dismembrator at maximum output ( ̃70%) with a half inch diameter probe (both available from Fisher Scientific, Suwanee, Ga.). The sonication disrupted bacterial cells and sheared DNA. In certain cases the lysate was then incubated overnight with stirring at 4° C. to ensure full dissolution of the recombinant protein—this was particularly useful for wild-type Ara h 1 and mutant Ara h 2. The lysate was then centrifuged at 27,000 g for 60 minutes (13,000 rpm in a Sorvall RC-5BTM centrifuge) to remove cellular debris. The supernatant was removed and re-centrifuged at 27,000 g for 30 minutes. Finally, the supernatant was filtered through a 0.45 μm membrane.
Column Preparation
Recombinant proteins were purified by means of column chromatography using a HIS•BIND® resin. As described above, all recombinant proteins had a 6×-His tag which binds to Ni2+ cations that are immobilized on the resin. A large column was packed with a settled bed volume of 25 ml of HIS•BIND® resin from Novagen, Madison, Wis. The binding capacity of the resin was estimated at 8 mg protein/ml using known amounts of 6×-His tagged β-galactosidase. The column was then washed with the following sequence of washes to charge and equilibrate the column (one volume is equivalent to the settled bed volume):

    
    
        (a) 75 ml (3 volumes) sterile deionized water
        (b) 125 ml (5 volumes) 1× charge buffer (50 mM NiSO4)
        (c) 75 ml (3 volumes) 1× binding buffer.

Purification

    
    


An open gravity flow system was used for purification. A flow rate of 250 ml per hour was used with a bed volume of 25 ml (or 10 volumes per hour). Purification included the following steps:

    
    
        (a) Binding buffer was allowed to drain to the top of column bed.
        (b) 600 ml of the prepared extract was loaded onto the column.
        (c) The column was washed with 300 ml (12 volumes) of 1× binding buffer.
        (d) The column was washed with 200 ml (8 volumes) of 1× binding buffer with 20 mM imidazole.
        (e) The column was washed with 150 ml (6 volumes) of 1× binding buffer.

Protein Refolding and Elution

    
    


After the column resin had been washed, a slow refolding step was included to allow the recombinant proteins to refold correctly and to increase the solubility of the final eluted purified protein. The recombinant proteins were refolded using a linear gradient of urea from 6 M down to 0 M in 1× refolding buffer (0.5 M NaCl, 20 mM Tris-HCl and 1 mM PMSF (phenylmethylsulfonyl fluoride) at pH 7.9) over a period of between 2 hours and overnight. The column flow system was closed for the refolding step and a model 750 gradient maker from Life Technologies, Bethesda, Md. was used for this purpose.
The refolded proteins were then eluted from the column using 1 M imidazole in an elution buffer (0.5 M NaCl, 20 mM Tris-HCl and 1 mM PMSF at pH 7.5). Typically, most of the protein could be recovered using 3×25 ml washes.
Processing the Purified Sample
The imidazole and other salts were removed by dialysis into 1×PBS using SPECTRA/POR® 7 dialysis membrane from Spectrum Laboratories, Rancho Dominguez, Calif. These membranes have a molecular weight cut-off of 3,500 kD. Typically, a 200 ml eluate was dialyzed in 4000 ml of 1×PBS for at least 2 hours at 4° C. The dialysis was then repeated with fresh 4000 ml of 1×PBS overnight, again at 4° C. The sample was then exchanged twice using an AMICONTM stirred-cell into 1×PBS with 1 mM PMSF as the exchange buffer at room temperature.
Example 5
Ara h 1 Mutant Protein with Reduced IgE Binding
5.1 Introduction
In order to modulate IgE reactivity of Ara h 1 a recombinant Ara h 1 protein was constructed with mutations in the immunodominant IgE binding epitopes (see MUT1 in Example 4, Section 4.2 and Table 8). The abilities of the wild-type and mutant recombinant Ara h 2 proteins to react with IgE were then tested in Western blot analysis with sera from peanut-sensitive individuals. As compared to wild-type Ara h 1, the mutant Ara h 1 protein bound less IgE in 50% of patients tested.
5.2 Materials and Methods
Recombinant wild-type and MUT1 versions of Ara h 1 were prepared as described in Example 4, Section 4.2. MUT1 includes a single alanine mutation in epitopes 1-6 and 17 as shown in Table 11.







TABLE 11







Mutations in MUT1 version of Ara h 1








Epitope
Mutation











1
K32A


2
D52A


3
V72A


4
R91A


5
D103A


6
R109A


17
R499A









A western blot control was performed on the wild-type and mutant Ara h 1 recombinant proteins to ensure that an equal amount of each protein was used in these studies. Equal amounts of wild-type and mutant Ara h 1 were detected and both proteins migrated at their expected molecular weights ( ̃65 kD).
5.3 Results
Western blots of wild-type and mutant recombinant proteins probed with individual peanut-sensitive patient sera were performed. The results are summarized in Table 12. Data for each patient is numbered 1-10 in the first column. The second column lists the epitopes that each patient recognized in the wild-type protein that were changed in the mutant protein. The third column lists the epitopes that each patient recognized in the wild-type protein that were not changed in the mutant protein. The fourth column shows the relative IgE binding affinity of the mutant protein vs. the wild-type protein. In 50% of individual cases IgE binding to the mutant protein was significantly reduced.







TABLE 12







Relative affinity of IgE to wild-type and mutant Ara h 1










Patient
Mutated epitopes
Wild-type epitopes
Relative binding













1
1, 4, 5, 17
8, 13
Decreased


2
2, 3, 4, 17
14, 18
Equal


3
4, 5, 17
11, 14, 18-20, 22
Increased


4
2, 4, 5, 17
9, 23
Decreased


5
1, 4, 17
9, 10, 12-15, 18, 21, 22
Equal


6
4, 17
8, 9, 20, 23
Decreased


7
1, 2, 4, 17
13
Equal


8
1, 3, 4, 17
13, 22
Equal


9
1, 2, 4, 17
10
Decreased


10
3, 17
8, 9, 10, 11
Decreased









5.4 Conclusion
These results indicate that it is possible to produce a mutated recombinant Ara h 1 protein that binds substantially lower amounts of serum IgE from peanut-sensitive patients.
Example 6
Ara h 2 Mutant Proteins with Reduced IgE Binding
6.1 Introduction
In order to modulate IgE reactivity of Ara h 2 a variety of recombinant Ara h 2 proteins were constructed with mutations in IgE binding epitopes (see MUT1, MUT2 and MUT3 in Example 4, Section 4.3 and Table 9). The abilities of the wild-type and mutant recombinant Ara h 2 proteins to react with IgE were then tested in Western blot analysis with sera from peanut-sensitive individuals. As compared to wild-type Ara h 2, the mutant Ara h 2 proteins bound less IgE, similar amounts of IgG, and exhibited a comparable ability to stimulate T-cell proliferation.
6.2 Materials and Methods
Recombinant wild-type and MUT1, MUT2 and MUT3 versions of Ara h 2 were prepared as described in Example 4, Section 4.3. The mutations of MUT1, MUT2 and MUT3 are shown in Table 13.







TABLE 13







Mutations in MUT1, MUT2 and MUT3 versions of Ara h 2











Epitope
Mutation
MUT1
MUT2
MUT3





1, 2
Q23A

X



1, 2
W25A


X


3
Q34A

X


3
E38A
X

X


4
P44A
X

X


5
D56A


X


6
D63A
X
X
X


7
D70A
X
X
X


8
R123A


X


9
L133A


X


10 
L150A


X









6.3 Results
IgE Binding to MUT1 and MUT3 vs. Wild-Type Ara h 2 Using Pooled Sera
Equal amounts of purified wild-type and mutant Ara h 2 proteins (MUT1 and MUT3) were separated by gradient (4-20%) PAGE and electrophoretically transferred onto nitrocellulose paper. The blots were incubated with antibody directed against N-terminal T7-tag or pooled serum from peanut-sensitive patients. While binding to the T7 tag remained relatively constant, IgE binding was dramatically decreased in the mutants.
IgE Binding to MUT1 and MUT3 vs. Wild-Type Ara h 2 Using Individual Sera
IgE binding to mutated recombinant Ara h 2 proteins (MUT1 and MUT3) as compared to the wild-type was then examined in Western blot analysis using individual patient sera. Laser densitometry was used to quantitate relative IgE binding. While IgE binding to MUT3 was dramatically reduced for each individual, some differences were observed between the different individuals in the group with MUT1.
Inhibition of IgE Binding to Native Ara h 2
To further characterize binding of IgE to MUT1 and MUT3, an inhibition binding assay was performed. 0.5 μg of the native Ara h 2 protein purified from crude peanut extract was loaded onto each member of a set of nitrocellulose membranes using a slot-blot apparatus. The membranes were then incubated with pooled patient serum (1:20) in the presence or absence of different concentrations of wild-type Ara h 2, MUT1, MUT3, and as controls rice protein or recombinant wild-type Ara h 1. Membranes were probed for bound IgE with 125I-labeled anti-human IgE antibody. Laser densitometry of the autoradiograms was used to quantitate the relative amounts of IgE binding. While MUT3 had a negligible effect (same as control) on IgE binding to native Ara h 2, MUT1 inhibited binding at similar levels as recombinant wild-type Ara h 2.
T-Cell Proliferation in Presence of MUT3
Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood of peanut-sensitive patients by density gradient centrifugation on Ficoll. 2×105 cells per well were incubated in triplicates for 7 days in RPMI media with 5% human AB serum in the presence of 10 μg/ml of the native Ara h 2 protein purified from the crude peanut extract or recombinant Ara h 2 proteins purified from E. coli. Cells incubated in media only were used as a control. Proliferation was measured by the incorporation of tritiated thymidine. The stimulation index (SI) was calculated as a ratio of radioactivity for the cells growing in the presence of allergen to that for the cells growing in media alone. Relatively low proliferation was observed in the presence of MUT3 suggesting that T-cell epitopes may be affected by mutagenesis of overlapping IgE epitopes.
MUT2 Binds Less IgE But Similar Amounts of IgG as Wild-Type Ara h 2
MUT2 includes mutations within IgE epitopes 3, 6, and 7 that were determined to be immunodominant in Example 2. MUT2 was produced and immunoblot analysis performed using serum from peanut-sensitive patients as described above. The results showed that MUT2 bound significantly less IgE than recombinant wild-type Ara h 2 but bound similar amounts of IgG.
MUT2 Retains the Ability to Activate T-Cell Proliferation
MUT2 was also used in T-cell proliferation assays to determine if it retained the ability to activate T-cells from peanut-sensitive individuals. Proliferation assays were performed on T-cell lines grown in short-term culture developed from six peanut-sensitive patients. T-cells lines were stimulated with either 50 μg of crude peanut extract, 10 μg of native Ara h 2, 10 μg of recombinant wild-type Ara h 2, or 10 μg of MUT2 and the amount of incorporated 3H-thymidine was determined for each cell line. Results were expressed as the average stimulation index (SI) which reflects the fold increase in 3H-thymidine incorporation exhibited by cells challenged with allergen when compared with media treated controls. MUT2 exhibited a comparable ability to stimulate T-cell proliferation as wild-type Ara h 2.
MUT2 Elicits a Smaller Wheal and Flare in Skin Prick Tests than Wild-Type Ara h 2
MUT2 and wild-type recombinant Ara h 2 were used in a skin prick test of a peanut-sensitive individual. 10 μg of these proteins were applied separately to the forearm of a peanut-sensitive individual, the skin pricked with a sterile needle, and 10 minutes later any wheal and flare that developed was measured. The wheal and flare produced by wild-type Ara h 2 (8 mm×7 mm) was approximately twice as large as that produced by MUT2 (4 mm×3 mm). A control subject (no peanut hypersensitivity) tested with the same proteins had no visible wheal and flare but, as expected, gave positive results when challenged with histamine. In addition, the test subject gave no positive results when tested with PBS alone. These results indicate that an allergen with only 50% of its IgE epitopes modified (i.e., 5/10) can give measurable reduction in reactivity in an in vivo test of a peanut-sensitive patient.
Example 7
In Vitro Safety Assay Using a Model Cell System for Mediator Release
7.1 Materials
Rat basophil leukemia cells (RBL-2H3) were “humanized” by transfection with the α chain of the human FcεRI receptor and thus enabled to bind human IgE. Crude peanut extract was prepared from Southeastern runners as described in Burks et al., J Allergy Clin. Immunol., 88:172, 1991. Crude soybean and crude pea extract were prepared using similar methods. Purified native Ara h 2 (nat Ara h 2) was prepared from crude peanut extract as described in Sen et al., J. Immunol. 169:882, 2002. “Wild-type” recombinant Ara h 2 (rAra h 2) was prepared as described in Example 4 above. “Mutant” recombinant Ara h 2 (mut Ara h 2) was prepared with the mutations of MUT3 that are shown in Table 9 of Example 4.
7.2 Methods
Transfectants were cultured in Eagle's MEM with 10% FCS, 0.1% geneticin sulfate, harvested in the stationary phase and transferred to 96-well microtiter plates (1.5×105 cells/well) for the mediator release assay as described elsewhere in Hoffmann et al., J. Allergy Clin. Immunol. 99:227, 1997. Deviating from this protocol, transfectants were passively sensitized by incubation with human serum IgE for 18 hours at 37° C. and 5% carbon dioxide. Dilutions of sera from three peanut allergic patients (JB, RW and PEI 163) were optimized by preliminary titrations (final serum dilution in 100 μl MEM were JB 1:100, RW 1:100 and PEI 163 1:80). After sensitization, the adherent cell layer was washed three times with Tyrode's buffer and incubated with 100 μl of serial dilutions of the cross-linking agents (nat Ara h 2, rAra h 2, mut Ara h 2, crude peanut extract, crude soybean extract, or crude pea extract) in Tyrode's buffer containing 50% D2O (Maeyama et al., J. Biol. Chem. 261:2583, 1986) for 1 hour at 37° C. For convenience, β-hexosaminidase was measured which has been shown to be released at the same rate as histamine (Schwartz et al., J. Immunol. 126:1290, 1981). To determine the enzymatic activity, 30 μl of the supernatant were transferred into a new microtiter plate and incubated with 50 μl of the substrate p-nitrophenyl-N-acetyl-β-D-glucosaminide (1.3 mg/ml in 0.1 M phosphate, 0.05 M citrate, pH 4.5) for 1 hour at 37° C. After addition of 100 μl 0.2 M glycin, pH 10.7, the absorbance was read at 405 nm (reference: 620 nm). As a consistency control for each microtiter plate, cells were sensitized with human myeloma IgE (hu IgE) (Biogenesis, Poole, UK: 1:5000) and stimulated with goat anti-human IgE (Nordic, Tilburg, NL: 1:1000). Spontaneous release (0%) was determined by omitting the cross-linking agents, the total enzyme content (100%) was measured by lysing the cells with 1% Triton X-100. Allergen-specific release was calculated as percent of total mediator content after correction for spontaneous release.
7.3 Results
FIGS. 10A-C compare the allergen-specific release levels that were measured when the IgE coated transfectant cells were exposed to a range of concentrations (0.001 to 1000 ng/ml) of the different cross-linking agents. For each patient (JB, RW and PEI 163), the level of release was lowest when the cells were exposed to crude soybean or pea extract and greatest when the cells were exposed to nat Ara h 2. In between, crude peanut extract caused greater release than rAra h 2 which in turn caused greater release than mut Ara h 2.
Example 8
Ara h 3 Mutant Protein with Reduced IgE Binding
8.1 Introduction
In order to modulate IgE reactivity of Ara h 3 a recombinant Ara h 3 protein was constructed with mutations in the immunodominant IgE binding epitopes (see MUT1 in Example 4, Section 4.4 and Table 10). The abilities of the wild-type and mutant recombinant Ara h 3 proteins to react with IgE were then tested in Western blot analysis with sera from peanut-sensitive individuals. As compared to wild-type Ara h 3, the mutant Ara h 3 protein bound less IgE.
8.2 Results
The proteins were probed with serum IgE from three patients previously shown to recognize recombinant Ara h 3. While wild-type Ara h 3 was bound by IgE, the mutated Ara h 3 protein was not recognized by serum IgE from the peanut-sensitive patients.
Example 9
In Vitro and In Vivo Assays with E. coli Cells Expressing WT Ara h 1-3
9.1 Methods for Killing E. coli Cells
Recombinant wild-type Ara h 1, Ara h 2, and Ara h 3 were produced in E. coli BL21 cells as described in Example 4. Several methods of killing the allergen-producing E. coli were then tested. As non-limiting examples, E. coli cells were killed by heat (at temperatures ranging from 37° C. to 95° C.), by using ethanol (0.1% to 10%), and by using solutions containing iodine (0.1% to 10%). Survival was determined by plating 100 μl of cells onto agar plates, and subsequently counting the resulting colonies. The most reproducible method was heat-killing with incubation at 60° C. for 20 minutes resulting in 100% death.
9.2 Production of Wild-Type Ara h 1-3
The amounts of each allergen that were produced by the E. coli cells were measured using an immunoblot assay that made use of the 6×-His tag present on each of the recombinant allergens Ara h 1-3. The amount of allergen produced on a per cell basis varied depending on which clone was tested. For this particular preparation, more Ara h 3 was produced than Ara h 2 and Ara h 1 (Ara h 3>Ara h 2>>Ara h1). Best estimates for the amount of allergen delivered in 100 μl inoculum of E. coli cells (O.D. of 2.0 at 600 nm) varied from about 1 μg of Ara h 1 to about 20 μg of Ara h 3.
9.3 Release of Ara h 1-3 from Heat-Killed E. coli Cells
In order to determine if the cells remained intact after heat-killing, the amount of allergen released into the media was measured. A dot-blot assay was developed that utilized as controls, purified recombinant allergens (see Example 4, Section 4.6) applied to a filter at known concentrations and serum IgE from peanut-sensitive patients. The assay detected and quantified the amount of allergen present in 100 μl of supernatant after pelleting heat-killed bacteria. The level of allergen released varied and was dependent on the expression vector and protein tested. In general, for this particular preparation, more Ara h 2 was released than Ara h 1 and Ara h 3 (Ara h 2>>Ara h 1>Ara h 3). As described previously, in certain embodiments of the invention, released allergen can be removed from an inventive composition using standard washing methods. For the purpose of these experiments the compositions were not washed.
9.4 Murine Immune Response to Heat-Killed E. coli Cells Expressing WT Ara h 1-3
The transformed cells were injected into C3H/HEJ mice to determine if the allergen-expressing E. coli elicited an immune response. The following protocol was utilized to assess the immune response. Blood was collected from the tail vein of each mouse used before the first injection. Enough blood was collected to perform an antibody ELISA for each allergen. On day 0 each mouse was injected with 100 μl of the heat-killed E. coli samples subcutaneously (sc) in the left hind flank. The mice were given a second boosting injection on day 14 using the same procedure. On day 21, a second blood sample was collected from each mouse. Blood samples at day 0 and day 21 were assayed for IgG1 and IgG2a antibodies to either Ara h 1, Ara h 2, or Ara h 3 by an ELISA assay.
Mice injected with E. coli producing Ara h 1 did not give detectable levels of any immunoglobulin to the Ara h 1 allergen. Without limitation to theory, it can be speculated that this may be due to the relatively small amounts of Ara h 1 produced by these cells (see Section 9.2). Mice injected with E. coli producing Ara h 2 contained relatively high levels of IgG1 and IgG2a. Again, without limitation to the cause, it can be speculated that this may be due to the amount of Ara h 2 released from these cells (see Section 9.3). Mice injected with E. coli producing Ara h 3 contained relatively high levels of IgG2a (indicative of a Th1-type response) and elicited relatively low levels of IgG1 (indicative of a Th2-type response). Overall, the data in this Example should be cautiously interpreted; however, the general trend suggests that more mice exhibited an IgG2a response than IgG1 response when the protein allergen was both expressed to a sufficient level and appropriately encapsulated within the heat-killed E. coli cells.
Example 10
In Vivo Safety Assay Using Mice Sensitized to Crude Peanut Extract
10.1 Materials
Female C3H/HeJ mice, 5 weeks of age were purchased from the Jackson Laboratory (Bar Harbor, Me.) and maintained on peanut-free chow, under specific pathogen-free conditions. Standard guidelines, Institute of Laboratory Animal Resources Commission of Life Sciences NRC, National Academy Press, 1996, for the care and use of animals were followed.
Crude peanut extract (CPE) was prepared from Southeastern runners as described in Burks et al., J. Allergy Clin. Immunol., 88:172, 1991. Purified native Ara h 1 was prepared from CPE as described in Maleki et al., J. Immunol. 164:5844, 2000. Purified native Ara h 2 was prepared from CPE as described in Sen et al., J. Immunol. 169:882, 2002. “Wild-type” Ara h 1, Ara h 2 and Ara h 3 allergens were prepared as described in Example 4 above. “Mutant” Ara h 1, Ara h 2 and Ara h 3 allergens were prepared with the mutations of MUT2 Ara h 1 (Table 8), MUT3 Ara h 2 (Table 9) and MUT1 Ara h 3 (Table 10) as described in Example 4 above. Heat-killed E. Coli expressing recombinant versions of Ara h 1, Ara h 2 and Ara h 3 were prepared by heating harvested E. coli cells to 60° C. for 20 minutes.
10.2 Methods
The sensitization and challenge protocols that were used in this Example are outlined in FIG. 11. All mice were sensitized by intraperitoneal (ip) injection of 500 μg CPE and 2 mg alum in 400 μl phosphate buffered saline (PBS) at weeks 0, 1 and 3.
Tail vein blood was obtained following sensitization (at week 4 or one day prior to challenge at week 5) to detect any potential bias caused by differences in the levels of CPE-specific IgE. Sera were collected and stored at −80° C. Levels of CPE-specific IgE were measured by ELISA as described in Li et al., J. Allergy Clin. Immunol. 106:150, 2000.
Five weeks following the initial sensitization, mice were challenged ip with a variety of compositions (see Results and FIG. 11). Anaphylactic symptoms were evaluated 30-40 minutes following challenge utilizing a 0-5 scoring system, modified slightly from previous reports (Li et al., J. Allergy Clin. Immunol. 103:206-214; Poulsen et al., Clin. Allergy; 17:449-458, 1987; McCaskill et al., Immunology, 51:669-677, 1984): 0=no symptoms; 1=scratching and rubbing around the nose and head; 2=puffiness around the eyes and mouth, diarrhea, pilar erecti, reduced activity and/or decreased activity with increased respiratory rate; 3=wheezing, labored respiration, cyanosis around the mouth and the tail; 4=no activity after prodding, or tremor and convulsion; and 5=death.
10.3 Results (G1-G5)
Five groups of five mice (G1-G5) were used in a first sensitization and challenge experiment. The individual and average IgE levels (ng/ml) measured one day prior to challenge at week 5 (i.e., post-sensitization) are compared in Table 14. Although there was some variability between individual mice in each group, the average IgE levels were comparable. The mice in each group were then challenged with an ip injection of one of three different compositions at week 5: G1 mice were challenged with CPE (600 μg); G2 mice were challenged with HKE-P123 (200 μg of each); G3 mice were challenged with HKE-MP123 (200 μg of each); G4 mice were challenged with P123 (200 μg of each); and G5 mice were challenged with MP123 (200 μg of each).
The individual and average symptom scores at week 5 for the five groups of mice are compared in Table 14. While mice challenged with CPE (G1) exhibited wheezing, labored respiration, cyanosis around the mouth and the tail and/or death after plasma collection, mice exposed to recombinant peanut allergens or HKE expressing these peanut allergens (G2-G5) exhibited mild diarrhea or no symptoms. The rectal temperatures of each of the mice were also measured at week 5 and are compared in Table 14.







TABLE 14







In vivo results for G1-G5









W 5












W0, W1, W3

W 51


Temp.


Sensitization
Mouse
IgE (ng/ml)
Challenge
Score
(° C.)















CPE
1
2403
CPE
3
36.0


(0.5 mg)
2
2555
(0.6 mg)
32
36.3



3
2510

32
35.7


G1
4
3133

32
36.0



5
2363

3
36.1



Average
2593

3.0
36.0


CPE
1
2272
HKE-P123
0
36.9


(0.5 mg)
2
3121
(0.2 mg each)
0
35.9



3
1936

0
36.7


G2
4
1487

0
35.3



5
1968

0
36.8



Average
2157

0.0
36.3


CPE
1
3157
HKE-MP123
23
36.0


(0.5 mg)
2
3358
(0.2 mg each)
0
36.4



3
2296

0
36.4


G3
4
2679

23
36.6



5
2515

0
36.3



Average
2801

0.8
36.3


CPE
1
2778
P123
0
38.0


(0.5 mg)
2
2651
(0.2 mg each)
0
37.9



3
2631

0
37.7


G4
4
2451

0
38.4



5
2093

0
38.2



Average
2521

0.0
38.0


CPE
1
2219
MP123
0
37.6


(0.5 mg)
2
1893
(0.2 mg each)
0
37.3



3
1642

0
37.5


G5
4
2420

0
37.5



5
2498

0
37.5



Average
2187

0.0
37.5





1IgE values were measured one day prior to challenge at week 5.


2Died after plasma collection.


3Diarrhea.






10.4 Results (G6-G10)

The sensitization and challenge experiments were repeated at a higher challenge dosage with five new groups (G6-G10) each including two sub-groups A and B of 2-5 mice. Again, the individual and average IgE levels (ng/ml) were measured one day prior to challenge at week 5 for the mice in each of the three groups and are compared in Table 15. Although there was some variability between individual mice in each group, the average IgE levels were again comparable between the five groups.
The mice were challenged with an ip injection of one of three different compositions at week 5: G6 mice were challenged with CPE (3 mg); G7 mice were challenged with HKE-MP123 (1 mg of each); G8 mice were challenged with HKE-MP123 (1 mg of each); G9 mice were challenged with P123 (1 mg of each); and G10 mice were challenged with MP123 (1 mg of each).
The individual and average symptom scores at week 5 for the five groups of mice are compared in Table 15. Again, while mice exposed to CPE (G6) exhibited wheezing, labored respiration, cyanosis around the mouth and the tail and/or death, mice exposed to recombinant peanut allergens or HKE expressing these peanut allergens (G7-G10) exhibited no symptoms. The rectal temperatures of each of the mice were also measured at week 5 and are compared in Table 15.







TABLE 15







In vivo results for G6-G10









W 5












W0, W1, W3

W 51


Temp.


Sensitization
Mouse
IgE (ng/ml)
Challenge
Score
(° C.)















CPE
1A
1799
CPE
3
31.8


(0.5 mg)
2A
1702
(3 mg)
3
32.0



3A
1956

5
31.3


G6
4A
2092

5
31.6



5A
1556

3
32.5



1B
1803

3
32.3



2B
1902

5
33.5



3B
1818

3
31.4



4B
2161

3
33.8



Average
1865

3.7
32.2


CPE
1A
1675
HKE-P123
0
34.8


(0.5 mg)
2A
1991
(1 mg each)
0
34.5



3A
1702

0
33.8


G7
4A
1479

0
34.0



1B
1640

0
34.1



2B
1819

0
33.8



3B
1591

0
34.4



4B
1710

0
34.0



Average
1701

0.0
34.2


CPE
1A
2019
HKE-MP123
0
35.6


(0.5 mg)
2A
1826
(1 mg each)
0
35.3



3A
2027

0
35.9


G8
4A
1883

0
36.4



1B
1990

0
36.2



2B
1648

0
35.4



3B
1354

0
35.3



4B
1536

0
35.9



Average
1786

0.0
35.8


CPE
1A
1891
P123
0
34.5


(0.5 mg)
2A
1726
(1 mg each)
0
35.5



3A
2287

0
36.3


G9
4A
1607

0
36.6



1B
1863

0
35.2



2B
2254

0
34.9



3B
1738

0
36.3



Average
1910

0.0
35.6


CPE
1A
1363
MP123
0
37.4


(0.5 mg)
2A
1485
(1 mg each)
0
36.6



3A
1669

0
37.0


G10
4A
1844

0
37.0



1B
1668

0
37.1



2B
1532

0
35.3



Average
1678

0.0
36.6





1IgE values were measured one day prior to challenge at week 5.






10.5 Results (G11-G14)

The sensitization and challenge experiments were repeated using a different set of challenge compositions with four more groups (G11-G14) each including 4-6 mice. The mice were challenged with an ip injection of one of three different compositions at week 5: G11 mice were challenged with CPE (3 mg); G12 mice were challenged with NP12 (1 mg of each); G13 mice were challenged with P123 (1 mg of each); and G14 mice were challenged with MP123 (1 mg of each).
The individual and average symptom scores at week 5 for the four groups of mice are compared in Table 16. While the mice exposed to CPE or purified native Ara h 1 and Ara h 2 (G11 and G12) exhibited severe anaphylactic symptoms (i.e., scores of 2-4), the mice that were exposed to wild-type peanut proteins (G13) exhibited mild reactions (i.e., symptom scores of 1-2) and the mice that were exposed to mutant peanut proteins (G14) exhibited no reactions. The rectal temperatures of each of the mice were also measured at week 5 and are compared in Table 16.







TABLE 16







In vivo results for G11-G14








W0, W1, W3
W 5











Sensitization
Mouse
Challenge
Score
Temp. (° C.)














CPE
1
CPE
2
33.4


(0.5 mg)
2
(3 mg)
3
32.3



3

3
32.1


G11
4

4
31.9



Average

3.0
32.4


CPE
1
NP12
4
31.5


(0.5 mg)
2
(1 mg each)
2
33.3



3

2
34.2


G12
4

2
32.8



5

2
34.9



Average

2.4
33.3


CPE
1
P123
2
34.4


(0.5 mg)
2
(1 mg each)
2
35.6



3

2
34.8


G13
4

1
34.5



5

1
36.2



6

1
35.5



Average

1.5
35.2


CPE
1
MP123
0
35.4


(0.5 mg)
2
(1 mg each)
0
36.1



3

0
36.2


G14
4

0
35.3



5

0
35.6



6

0
36.8



Average

0.0
35.9









Example 11
In Vivo Experiments Testing Different Delivery Routes for Desensitization
11.1 Introduction
The sensitization, desensitization and challenge protocols that were used in this Example are outlined in FIG. 12. Ten groups of mice (G1-G10) were used for these in vivo desensitization experiments. The 5 week old female C3H/HeJ mice (approx. 10 per group) were first sensitized with crude peanut extract and cholera toxin (CT) over a period of 8 weeks (W0-W8). Mice were deprived of food for 2 hours and given 300 μl of 1.5% NaHCO3 30 minutes before feeding to neutralize stomach acid. Sensitization was then performed by intragastric (ig) administration of 10 mg of crude peanut extract (CPE) together with 20 μg of CT on day 0 (W0), then boosted weekly for 6 weeks (W1-W6) and again at week 8 (W8).
The mice were then treated according to ten different desensitization protocols at weeks 10, 11, and 12 (W10-W12). Finally the mice were challenged intragastrically (ig) with crude peanut extract at week 13 (W13). G1 mice were sham desensitized at weeks 10-12, i.e., treated with a placebo. G2, G3, and G4 mice were desensitized via the subcutaneous (sc) route with HKE-MP123 (30, 15, and 5 μg of each, respectively). G5 mice were desensitized via the intragastric (ig) route with HKE-MP123 (50 μg of each). G6 mice were desensitized via the rectal (pr) route with HKE-MP123 (30 μg of each). G7 mice were desensitized via the rectal (pr) route with MP123 alone (30 μg of each). G8 mice were naïve, i.e., were not sensitized with crude peanut extract and CT during weeks 0-8 and received no desensitization treatment. G9 mice were desensitized via the subcutaneous (sc) route with heat-killed L. monocytogenes (HKL) alone. G10 mice were desensitized via the subcutaneous (sc) route with heat-killed L. monocytogenes expressing mutated Ara h 1-3 (HKL-MP123, 30 μg of each).
11.2 Results
The average IgE levels (ng/ml) at weeks 3, 8, 12, and 14 for the ten groups of mice (G1-G10) are shown in FIG. 13. As compared to the sham desensitized mice, the increase in IgE levels during the desensitization period (W10-W12) was dramatically reduced in all the desensitized groups except for the mice that were treated with HKL alone (G9) or via the ig route with HKE-MP123 (G5).
The individual (symbols) and average (solid line) symptom scores (0-5) at week 14 for the ten groups of mice are compared in FIGS. 14 and 15. The improvement in symptom scores parallel the IgE data with dramatic improvements (from an average score of 3.5 to average scores of 0.4 or less) except for the groups of mice that were treated with HKL only (average score of 3.4) or via the ig route with HKE-MP123 (average score of 3.0).
The individual (symbols) and average (solid line) body temperatures (° C.) at week 14 for the ten groups of mice are compared in FIGS. 16 and 17. The trend in average body temperature correlates well with the results in FIGS. 13-15. In all treated groups the average body temperature at week 14 is higher than in the sham sensitized group. However, the increase is smallest for the group of mice that were treated with HKL only or via the ig route with HKE-MP123.
The individual (symbols) and average (solid line) airway responses (peak expiratory flow in ml/min) at week 14 for the ten groups of mice are compared in FIGS. 18 and 19. Peak flow values are dramatically improved in most groups except for the group of mice that were treated with HKL only or via the ig route with HKE-MP123.
FIGS. 20, 21, 22, and 23 compare the plasma histamine (nM), IL-4 (pg/ml), IL-5 (pg/ml), and IFN-γ (pg/ml) concentrations at week 14 for the ten groups of mice (G1-G10).
These experiments suggest that parenteral (e.g., subcutaneous) or rectal delivery of HKE-MP123 produce the best desensitization results. In this context, the inventors have also observed that subcutaneous delivery induces local skin inflammation. In contrast, rectal delivery does not induce local inflammatory reactions (see Example 12, Section 12.3).
Example 12
In Vivo Experiments Testing Different Compositions for Desensitization
This Example refers to various patents, publications, books, articles, and other references that are listed under Section 12.6. The contents of all of these items are hereby incorporated by reference in their entirety.
12.1 Introduction
This Example compares the efficacy of rectally administered HKE-MP123 and MP123 in the treatment of peanut allergy. Peanut allergic C3H/HeJ mice received HKE-MP123 weekly (9 or 90 μg) for 3 weeks or MP123 three times a week (9 or 90 μg) for 4 weeks. Following peanut challenge, anaphylactic symptom scores and plasma histamine levels were determined. Peanut-specific IgE and IgG2a levels were monitored. The effect of treatment on Th1/Th2 cytokine secretion by splenocytes (SPCs) was also determined. HKE-MP123 treatment at both 9 and 90 μg significantly reduced anaphylactic symptom scores, plasma histamine levels and serum peanut-specific IgE levels and increased IgG2a levels as compared to the sham-treated group. Overall, HKE-MP123 treatment was more effective than MP123 treatment. HKE-MP123 treatment significantly reduced IL-4 and IL-5, and increased IFN-γ secretion by SPCs. HKE-MP123 also decreased IL-10 and increased TGF-β. Rectal administration of HKE-MP123 did not cause local mucosal inflammation. This Example confirms that rectally administered HKE-MP123 significantly suppresses peanut allergy.
12.2 Materials
Five-week-old female C3H/HeJ mice were purchased from the Jackson Laboratory (Bar Harbor, Me.) and maintained on peanut-free chow under specific pathogen-free conditions. Standard guidelines for the care and use of animals were followed (see Ref. 15).
Freshly ground whole peanut, and crude peanut extract (CPE) were prepared as described in Ref 16 and employed as antigens. Cholera toxin (CT) was purchased from List Biological Laboratories, Inc. (Campbell, Calif.). Concanavalin A (Con A), and albumin-dinitrophenyl (DNP-albumin) were purchased from Sigma (St. Louis, Mo.). Antibodies for ELISAs (sheep anti-mouse IgE, and biotinylated donkey anti sheep IgG) were purchased from the Binding Site, Inc. (San Diego, Calif.). Anti-DNP IgE and IgG2a were purchased from Accurate Scientific, Inc. (Westbury, N.Y.).
12.3 Methods
Preparation of HKE-MP123 and MP123
E. coli BL21 clones expressing mutated Ara h 1 (MUT2, Table 8), Ara h 2 (MUT3, Table 9), and Ara h 3 (MUT1, Table 10), or carrying the pET24(a)+ vector alone, were generated as described previously in Example 4 (see also Refs. 10-11). The bacteria were grown and protein expression was induced following the protocol described in Ref 17. The bacteria were inactivated by incubation in a water bath at 65° C. for 30 minutes, cooled on ice for  ̃20 minutes and collected by centrifugation at 4,000 g at 4° C. for 30 minutes. The cell pellet was washed with ice-cold phosphate-buffered saline (PBS), centrifuged as above, and resupended in the same buffer. The final HKE-MP123 mixture containing approximately equal quantities of the three modified peanut proteins was prepared according to the specific recombinant protein content of the bacteria. Specific protein expression levels were determined by Western immunoblot analysis using serial dilutions of each individual HKE and known quantities of the purified recombinant protein as standards. In the vector-control HKE suspension (HKE-V), the total number of bacteria, measured as optimal unit (O.U.) at 600 nm, was matched to that in the high dose HKE-MP123. The effectiveness of the heat-inactivation procedure was determined by plating bacterial aliquots taken from the induced culture and the final HKE suspensions on a LB-agar plate containing 30 μg/ml of kanamycin. Less than 0.02% of the HKE bacterial cells were found to be viable. The integrity of the heat-treated bacteria was verified by Western immunoblot analysis of the specific protein content in the bacterial cell pellet and in the culture supernatant from each individual HKE obtained by centrifugation at 4,000 g for 2 minutes at room temperature. More than 99% of the specific recombinant protein was shown to be present in the bacterial cell pellet fraction.
The recombinant mutated Ara h 1, Ara h 2, and Ara h 3 proteins were purified using a HIS•BIND® Ni2+-chelating resin (Novagen, Madison, Wis.) according to the protocol described in Example 4 (see also Ref 10). Briefly, 6 to 12 liters of bacteria expressing one of the proteins were grown and induced as above. Bacteria were harvested and sonicated on ice for 20 minutes. The lysate was cleared and loaded on a chromatography column with  ̃25 ml of the HIS•BIND® resin loaded with Ni2+. The column was washed with the binding buffer, the bound protein was renatured using a linear gradient of urea from 6 M to 0 M in the binding buffer. The column was washed with the binding buffer, pH 7.5, and the protein was eluted with a linear gradient of 0 M to 1 M of imidazole in 6 bed volumes of the same buffer. The eluent ( ̃200 ml) was collected and dialyzed against two changes of 20 volumes each of PBS with 1 mM of phenylmethanesulfonyl fluoride (PMSF) at 4° C., for up to 20 hours total. The dialyzed protein was centrifuged at 23,000 g, 4° C. for 20 minutes and concentrated to an appropriate concentration using the AMICON® 8200 Stirred Cell with the YM-10 ultrafiltration membrane (Millipore, Billerica, Mass.). The protein concentration was determined by Micro BCATM Protein Assay (Pierce, Rockford, Ill.). The purity of the protein was checked by SDS gel-electrophoresis.
Intragastric Antigen-Sensitization, Challenge, and HKE-MP123 Treatment
Peanut sensitization and challenge followed the protocol described in Ref 18 and is outlined in FIG. 24. Briefly, mice were deprived of food for 2 hours. Sensitization was then performed by intragastric (ig) administration of 10 mg freshly ground whole peanut together with 20 μg of CT mixed with 300 μl of 1.5% NaHCO3. Mice were then boosted weekly for 6 weeks and again at week 8. Treatment began at week 10. Mice were treated with HKE-MP123 (9 or 90 μg), weekly for 3 weeks (G2 and G3), or with MP123 (9 or 90 μg), three times a week for 4 weeks (G4 and G5). Sham (saline)-treated (G1) and naïve (G8) mice were included as controls. Rectal administration was performed using an 18 gage catheter and 90 μl of each preparation was instilled while the mice were anesthetized with a mixture of ketamine and xylazine (80 mg/kg and 10 mg/kg). The catheter was inserted approximately 1.5 cm. All mice were challenged intragastrically at week 14 with peanut (50 mg/mouse) in 2 divided doses at 30-40 minutes intervals.
Assessment of Hypersensitivity Reactions
Anaphylactic symptoms were evaluated 30-40 minutes after the second challenge dose utilizing the scoring system described in Example 10, Section 10.2 (see also Refs. 14 and 16): 0=no symptoms; 1=scratching and rubbing around the snout and head; 2=puffiness around the eyes and snout, diarrhea, pilar erecti, reduced activity, and/or decreased activity with increased respiratory rate; 3=wheezing, labored respiration, cyanosis around the mouth and the tail; 4=no activity after prodding, or tremor and convulsion; 5=death. Scoring of symptoms was performed in a blinded manner.
Measurement of Plasma Histamine Levels
Plasma histamine levels in blood samples collected 30 minutes after the second ig challenge dose were determined using an enzyme immunoassay kit (ImmunoTECH, Inc., Marseille, France) as described by the manufacturer (see Ref. 19).
Measurement of Serum Peanut Specific IgE and IgG2a
Tail vein blood was obtained during sensitization/boosting, 1 day before treatment and 1 day prior to challenge. Sera were collected and stored at −80° C. Levels of peanut-specific IgE and IgG2a were determined as described in Refs. 14, 16 and 20. Briefly, plates were coated with CPE incubated overnight at 4° C., and then blocked and washed. Samples (1:10 dilutions for IgE and 1:50 for IgG2a) were added to the plates and incubated overnight at 4° C. and plates were then washed. For detecting IgE antibodies, sheep anti-mouse IgE (0.3 μg/ml) was added and incubated for 1 hour and after washing, biotinylated donkey anti-sheep IgG (0.5 μg/ml) was added and incubated at room temperature (RT) for 1 hour. After appropriate washing, avidin-peroxidase was added for an additional 15 minutes at room temperature. The reactions were developed with ABTS (KPL) and read at 405 nm. For IgG2a measurement, biotinylated rat anti-mouse IgG2a monoclonal antibodies (0.25 μg/ml) were used as the detection antibodies. Subsequent steps were the same as those for IgE measurement. Equivalent concentrations of peanut-specific IgE and IgG2a were calculated by comparison with a reference curve generated with anti-DNP IgE and IgG2a mouse monoclonal antibodies, as described in Refs. 16 and 21.
Cell Culture and Cytokine Measurements
SPCs were isolated from pooled spleens removed from each group of mice, which were sacrificed immediately after evaluation of the anaphylactic reactions, and cultured in RPMI 1640 containing 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% glutamine. SPCs were cultured in 24 well plates (4×106/well/ml) in the presence or absence of CPE (50 μg/ml) or Con A (2 μg/ml). Supernatants were collected after 72 hours of culture and aliquots were stored at −80° C. until analyzed. IL-4, IL-5 and IFN-γ levels were determined by ELISA according to the manufacturer's instructions.
Histology
In a preliminary study, we localized the distribution of rectally administered fluid by injection of 90 μl of 0.5% of Evans blue following the procedure described above, and found that the fluid entered the sigmoid colon. To determine whether rectal administration of HKE-MP123 causes local inflammation, we collected rectum and colon samples from HKE-MP123, and saline treated mice 24 hours following the initial rectal administration as well as following peanut challenge at week 14. Tissues were fixed in 10% neutral buffered formalin and 5-micrometer paraffin sections were stained with hematoxylin and eosin (H and E) and examined by light microscopy.
Statistical Analysis
Data were analyzed using SigmaStat statistical software package (SPSS Inc. Chicago, Ill.). For histamine, IgE, and cytokine levels, the differences between the groups were analyzed by One way ANOVA followed by the Bonferroni's t test for all pairwise comparisons, if the data passed normality testing. For symptom scores, the differences between the groups were analyzed by Kruskal-Wallis One Way Analysis of Variance on Ranks followed by all pairwise comparison procedure (Dunn's), if the data failed to pass the normality test. We computed N, the required sample size per group, for 80% power, using a two tail test at the 0.05 level based on our preliminary study; 5 mice per group are required. p values <0.05 were considered to be of statistical significance.
12.4 Results
HKE-MP123 is More Effective than MP123 in Preventing Peanut Hypersensitivity Reactions
Since anaphylactic reactions are the hallmark of peanut allergy, we first determined anaphylactic symptom scores 30 minutes following peanut-challenge. The severity of symptom scores in both low and high doses of HKE-MP123-treated groups were significantly reduced as compared with the sham treated group (FIG. 25, p<0.001). On the other hand, the symptom scores in low dose MP123-treated group were essentially the same as the sham-treated group. Although there was some reduction of symptom scores in the high dose MP123-treated group as compared with the sham-treated group, the reduction in this group did not reach statistical significance (p=0.065). These results demonstrate that HKE-MP123 is more effective than MP123 in protecting peanut-allergic mice from peanut-induced anaphylactic reactions. These results were obtained despite administering 9 more doses of MP123 of the same dosage (G4) and even at a dose that was 10 times higher (G5). In addition, treatment with HKE carrying vector alone showed some protective effect, but much less than the HKE-MP123 treatment (data not shown).
HKE-MP123 is More Effective than MP123 in Reducing Histamine Release
Histamine is one of the major mediators associated with anaphylactic reactions. To determine whether the protection against anaphylactic reactions in this model was associated with reduction of histamine release, we measured plasma histamine levels following challenge. We found that histamine levels were markedly reduced in both HKE-MP123 treated groups, being lowest in the high dose treated group (FIG. 26, p<0.05 and 0.001 respectively). However, histamine levels in the low and high dose MP123 treated-groups were not significantly different than the sham-treated group. These results support the clinical findings that HKE-MP123 was more effective than MP123 in protecting peanut-sensitized mice.
HKE-MP123 is More Effective than MP123 in Decreasing IgE and Increasing IgG2a Production
At week 10 following peanut-sensitization and prior to treatment, peanut-specific IgE levels were markedly elevated in all sensitized groups and were similar in each group. However, IgE levels in both HKE-MP123-treated groups were significantly lower than the sham-treated group at the time of challenge (week 14) (FIG. 27, p<0.001). The high dose MP123 treated group, but not the low dose MP123-treated group, also showed significantly lower IgE levels than sham-treated group (p<0.05), but significantly higher levels than HKE-MP123 treated groups (p<0.01 and 0.001vs. HKE-MP123 at 9 μg and 90 μg, respectively). Peanut-specific IgG2a levels were significantly increased in both HKE-MP123-treated groups (p<0.001) compared to the sham-treated group. However, no significant increase in IgG2a levels was seen in either low or high MP123-treated groups. These results demonstrate that HKE-MP123 treatment is more effective in suppressing IgE and enhancing IgG2 than MP123 protein treatment alone. Although the high dose of MP123 also significantly reduced IgE, the reduction was not accompanied by a protective effect against anaphylactic reactions.
HKE-MP123 is More Effective than MP123 in Modulating Th1, Th2 and T Regulatory Responses
It has been suggested that IFN-γ plays a role in the induction of oral tolerance (see Ref. 22) and that peanut allergy is a Th2-driven immune response (see Ref 23). IFN-γ, IL-4, IL-5 and IL-13 levels were therefore measured in SPC cell culture supernatants from each group of mice. IFN-γ levels were significantly higher in both HKE-MP123-treated groups compared to the sham-treated group (see FIG. 28A. p<0.05 and 0.001, respectively). However, there was no significant difference between the MP123-treated groups and the sham-treated group. IL-4, IL-5 and IL-13 levels were significantly reduced in SPC cultures from high dose HKE-MP123-treated groups compared to SPC cultures from sham-treated mice (see FIGS. 28B, C, and D. p<0.001, 0.01, and 0.001 respectively), and IL-4 and IL-13 levels were significantly reduced in SPC cultures from low dose HKE-MP123-treated groups compared to SPC cultures from sham-treated mice (p<0.001 and 0.01, respectively). On the other hand, IL-4, IL-5 and IL-13 levels were not significantly reduced in the low dose MP123-treated group compared to SPC cultures from sham-treated mice. Although IL-4 and IL-13 levels in the high dose MP123-treated group were also significantly lower than that in SPC cultures from sham-treated mice (p<0.01 and 0.05, respectively), they were significantly higher than that in cultures from the high dose HKE-MP123-treated mice (p<0.01 and 0.05 respectively).
We also measured TGF-β, a T regulatory cytokine and IL-10, a T suppressor cytokine, which are thought to be important in the development of oral tolerance (see Refs. 24-26). We found that IL-10 was reduced in both HKE-MP123-treated groups as compared with the sham-treated group, being lowest in the HKE-MP123 high dose treated group (see FIG. 28E. p<0.05 and 0.01, respectively). In contrast, TGF-β levels were significantly increased in the HKE-MP123-treated groups in a dose dependent manner (see FIG. 28F. p<0.05 and 0.001, respectively). MP123 treatment, regardless of the dose, did not produce any effect on either IL-10 or TGF-β production. These results indicate that HKE-MP123 had a broad immunoregulatory effect on Th1, Th2 and T regulatory cytokines and was more effective than MP123 alone.
HKE-MP123 Did not Induce a Local Inflammatory Reaction
Sigmoid colons were collected from peanut sensitized mice 24 hours after the final treatment dosage (FIG. 29A-C) and following peanut challenge (FIG. 29D-F). FIG. 29 compares the colons of sham treated mice (Panels A and D), HKE-MP123-treated mice (90 μg, Panels B and E), and naïve mice (Panels C and F). No inflammation was seen in any histologic sections. In addition, histological analyses of sigmoid colons from the MP123-treated group also showed no evidence of inflammation (data not shown).
12.5 Discussion
Peanut-induced anaphylaxis is an IgE mediated type I hypersensitivity, and histamine is one of the major mediators released by mast cells/basophils, which is at least in part responsible for provoking symptoms of anaphylaxis. In the present Example, it has been demonstrated that the rectal administration of HKE containing mutated peanut proteins significantly desensitized peanut allergic mice, as shown by a reduction of peanut-specific IgE levels, post-challenge histamine levels and symptom scores. On the other hand, treatment with MP123 alone, even with nine additional treatments, did not provide significant protection even though there was a moderate reduction in peanut-specific IgE. These results demonstrated that HKE-MP123 is more effective in protecting against anaphylaxis than MP123. The precise mechanisms underlying the enhanced potency of HKE-MP123 in desensitizing peanut allergy are unknown, although it is likely mediated by HKE's adjuvant effect since administering the purified protein alone did not provide significant protection.
In addition to efficacy, HKE-MP123 has several other benefits as a novel immunotherapeutic approach for the treatment of peanut allergy. First, since the engineered recombinant peanut proteins are generated in E. coli, using HKE-MP123 eliminates the need to purify the recombinant peanut proteins from E. coli and therefore is technically easier and less costly. Second, the E. coli organisms are still intact after heat killing and encapsulate the peanut proteins within the organism which prevents them from activating mast cells or basophils, resulting in an additional level of safety. Lastly, since the HKE-MP123 is administered into an environment replete with E. coli and other bacteria, there should be little concern about the safety of such vaccine administration. In this context, no evidence of inflammatory reactions was found at the immunization site and no signs of anaphylactic symptoms were observed during the desensitization phase.
It has been suggested that tolerance to food antigens induced via the gut involves IFN-γ (see Refs. 22, 27 and 28). According to the hygiene hypothesis, the increasing incidence of allergy in Westernized societies over the last decades (see Refs. 29-30) may, to some extent, be explained by a reduced microbial load early in infancy (see Refs. 30-32) which results in too little Th1 cell activity, and therefore insufficient IFN-γ to optimally cross-regulate Th2 responses (see Ref 33). A recent study suggests that peanut allergic status is characterized by a Th2 response whereas a Th1-skewed response underlies oral tolerance (see Refs. 23). We recently found that impaired induction of IFN-γ following oral antigen sensitization is associated with the susceptibility of C3H/HeJ mice to both milk allergy and peanut allergy (see Refs. 17 and 34). It is suggested that the goal of allergen-based immunotherapy is reestablishment of immunologic tolerance to allergen by redirecting T-cell immune responses from a Th2-skewed response to a more balanced Th1/Th2 response (see Ref. 35). In this Example, we found both high and low doses of HKE-MP123 induced significant increases in IFN-γ levels and reduced Th2 cytokine levels. This effect was associated with an increase of peanut-specific IgG2a and a reduction of peanut-specific IgE. Therefore, induction of IFN-γ by HKE-MP123 may play an important role in the suppression of Th2 cytokines and the reestablishment of oral tolerance to peanuts in this model.
IL-10, initially characterized as a Th2 cytokine (see Ref 36) which suppressed IFN-γ and IL-12 secretion (see Ref 37) and inflammatory responses in autoimmune diseases (see Ref 38), has been recently suggested to be important in the suppression of allergic inflammation (see Ref. 39). A recent study showed induction of IL-10+ CD25+ T-cells by grass pollen immunotherapy (see Ref 40). However, there are conflicting findings regarding the role of IL-10 in immunotherapy and a protective role of IL-10 in food allergy has not been established. We found that IL-10 levels were significantly increased in peanut allergic mice, which was associated with the induction of Th2 cytokines and reduction of Th1 cytokines (see Ref 41). Previous studies including ours showed that heat-killed L. monocytogenes immunotherapy-mediated protection against OVA-induced allergic airway responses and peanut-induced anaphylaxis in mice was associated with reduction of IL-10 (see Refs. 13 and 18). In the present Example, we found that IL-10 levels were reduced in both HKE-MP123-treated groups as compared with the sham-treated group, being lowest in the high dose HKE-MP123-treated group. These results suggest that IL-10 is unlikely to play a beneficial role in the HKE-MP123-mediated protective effect on peanut allergy.
TGF-β is suggested to be important in the development of oral tolerance to food allergens (see Ref. 26). Colostrum TGF-β concentrations were found to be lower in samples from mothers of infants with IgE mediated cow milk allergy than in samples from mothers of infants with non-IgE mediated cow milk allergy (see Ref 42). However, a relationship between allergen immunotherapy-mediated regeneration of oral tolerance to food antigen and TGF-β has not been demonstrated. In this study, we found that TGF-β levels were significantly increased in both HKE-MP123-treated groups, but not the MP123-treated group, and appeared to be dose dependent. These results taken together, suggest that IFN-γ and TGF-β might be important cytokines responsible for a HKE-MP123 mediated therapeutic effect in peanut allergy.
In conclusion, this Example demonstrates that the rectal administration of HKE-MP123 markedly reduces peanut specific-IgE and plasma histamine levels in peanut allergic mice and protects against systemic anaphylaxis. These effects are more effective than administering MP123 alone. The precise mechanisms associated with protection are not fully understood, but the results suggest that the protective effect may be a consequence of down-regulation of Th2 cytokines perhaps due to induction of IFN-γ and/or TGF-β by an HKE adjuvant effect.
12.6 References


    1. Sampson, H. A. Food allergy. Part 1: immunopathogenesis and clinical disorders. J. Allergy Clin. Immunol. 1999; 103:717.
    2. Yocum, M. W., J. H. Butterfield, J. S. Klein, G. W. Volcheck, D. R. Schroeder, and M. D. Silverstein. Epidemiology of anaphylaxis in Olmsted County: A population-based study. J. Allergy Clin. Immunol. 1999; 104:452.
    3. Sampson, H. A., L. Mendelson, and J. P. Rosen. Fatal and near-fatal anaphylactic reactions to food in children and adolescents. N. Engl. J. Med. 1992; 327:380.
    4. Bock, S. A., A. Munoz-Furlong, and H. A. Sampson. Fatalities due to anaphylactic reactions to foods. J. Allergy Clin. Immunol. 2001; 107:191.
    5. Sicherer, S. H., A. Munoz-Furlong, A. W. Burks, and H. A. Sampson. Prevalence of peanut and tree nut allergy in the US determined by a random digit dial telephone survey. J. Allergy Clin. Immunol. 1999; 103:559.
    6. Bock, S. A. The natural history of food sensitivity. J. Allergy Clin. Immunol. 1982; 69:173.
    7. Oppenheimer, J. J., H. S. Nelson, S. A. Bock, F. Christensen, and D. Y. Leung. Treatment of peanut allergy with rush immunotherapy. J. Allergy Clin. Immunol. 1992; 90:256.
    8. Leung, D. Y., H. A. Sampson, J. W. Yunginger, A. W. J. Burks, L. C. Schneider, C. H. Wortel, F. M. Davis, J. D. Hyun, and W. R. J. Shanahan. Effect of anti-IgE therapy in patients with peanut allergy. N. Engl. J. Med. 2003; 348:986.
    9. Li X. M. and H. A. Sampson. Novel approaches for the treatment of food allergy. Current Opinion in Allergy and Clinical Immunology 2002; 2:273.
    10. Burks, A. W., N. King, and G. A. Bannon. Modification of a major peanut allergen leads to loss of IgE binding. Int. Arch. Allergy Immunol. 1999; 118:313.
    11. Bannon, G. A., G. Cockrell, C. Connaughton, C. M. West, R. Helm, J. S. Stanley, N. King, P. Rabjohn, H. A. Sampson, and A. W. Burks. Engineering, characterization and in vitro efficacy of the major peanut allergens for use in immunotherapy. Int. Arch. Allergy Immunol. 2001; 124:70.
    12. Yeung, V. P., R. S. Gieni, D. T. Umetsu, and R. H. DeKruyff. Heat-killed Listeria monocytogenes as an adjuvant converts established murine Th2-dominated immune responses into Th1-dominated responses. J. Immunol. 1998; 161:4146.
    13. Hansen, G., V. P. Yeung, G. Berry, D. T. Umetsu, and R. H. DeKruyff. Vaccination with heat-killed Listeria as adjuvant reverses established allergen-induced airway hyperreactivity and inflammation: role of CD8+ T-cells and IL-18. J. Immunol. 2000; 164:223.
    14. Li, X. M., K. Srivastava, J. W. Huleatt, K. Bottomly, A. W. Burks, and H. A. Sampson. Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model. J. Immunol. 2003; 170:3289.
    15. Institute of Laboratory Animal Resources Commission of Life Sciences, N. R. C. 1996. Guide for the Care and Use of Laboratory Animals. National Academy Press.
    16. Li, X. M., D. Serebrisky, S. Y. Lee, C. K. Huang, L. Bardina, B. H. Schofield, J. S. Stanley, A. W. Burks, G. A. Bannon, and H. A. Sampson. A murine model of peanut anaphylaxis: T- and B-cell responses to a major peanut allergen mimic human responses. J. Allergy Clin. Immunol. 2000; 106:150.
    17. Burks, A. W., N. King, and G. A. Bannon. Modification of a major peanut allergen leads to loss of IgE binding. Int. Arch. Allergy Immunol. 1999; 118:313.
    18. Li, X. M., K. Srivastava, J. W. Huleatt, K. Bottomly, A. W. Burks, and H. A. Sampson. Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model. J. Immunol. 2003; 170:3289.
    19. Li, X. M., B. H. Schofield, C. K. Huang, G. A. Kleiner, and H. A. Sampson. A Murine Model of IgE Mediated Cow Milk Hypersensitivity. J. Allergy Clin. Immunol. 1999; 103:206.
    20. Lee, S. Y., C. K. Huang, T. F. Zhang, B. H. Schofield, A. W. Burks, G. A. Bannon, H. A. Sampson, and X. M. Li. Oral Administration of IL-12 Suppresses Anaphylactic Reactions in a Murine Model of Peanut Hypersensitivity. Clin. Immunol. 2001; 101:220.
    21. Lee, S. Y., C. K. Huang, T. F. Zhang, B. H. Schofield, A. W. Burks, G. A. Bannon, H. A. Sampson, and X. M. Li. Oral Administration of IL-12 Suppresses Anaphylactic Reactions in a Murine Model of Peanut Hypersensitivity. Clin. Immunol. 2001; 101:220.
    22. Husby, S. Sensitization and tolerance. Curr. Opin. Allergy Clin. Immunol. 2001; 1:237.
    23. Turcanu, V., S. J. Maleki, and G. Lack. Characterization of lymphocyte responses to peanuts in normal children, peanut-allergic children, and allergic children who acquired tolerance to peanuts. J. Clin. Invest. 2003; 111:1065.
    24. Strobel, S. Oral tolerance, systemic immunoregulation, and autoimmunity. Ann. N Y Acad. Sci. 2002; 958:47.
    25. Barnes, P. J. IL-10: a key regulator of allergic disease. Clin. Exp. Allergy 2001; 31:667.
    26. Husby, S. Sensitization and tolerance. Curr. Opin. Allergy Clin. Immunol. 2001; 1:237.
    27. Weiner, H. L. Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol. Today 1997; 18:335.
    28. Strobel, S. and A. M. Mowat. Immune responses to dietary antigens: oral tolerance. Immunol. Today 1998; 19:173.
    29. International Study of Asthma and Allergies in Childhood Steering Committee. Worldwide variation in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis and atopic eczema: ISAAC. Lancet 1998; 351:1225.
    30. von Mutius, E. The environmental predictors of allergic disease. J. Allergy Clin. Immunol. 2000; 105:9.
    31. Rook, G. A. and J. L. Stanford. Give us this day our daily germs. Immunol Today 1998; 19:113.
    32. Erb, K. J. Atopic disorders: a default pathway in the absence of infection? Immunol. Today 1999; 20:317.
    33. Brandtzaeg, P. Current Understanding of Gastrointestinal Immunoregulation and Its Relation to Food Allergy. Ann. N Y Acad. Sci. 2002; 964:13.
    34. Morafo, V., K. Srivastava, C. K. Huang, G. Kleiner, S. Y. Lee, Sampson H A, and Li X. M. 2003. Genetic susceptibility to food allergy is linked to differential TH2-TH1 responses in C3H/HeJ and BALB/c mice. J. Allergy Clin. Immunol. 111:1122.
    35. Durham, S. R. and S. J. Till. Immunologic changes associated with allergen immunotherapy. J. Allergy Clin. Immunol. 1998; 102:157.
    36. Fiorentino, D. F., M. W. Bond, and T. R. Mosmann. Two types of mouse T helper cell. IV. Th2 clones secrete a factor that inhibits cytokine production by Th1 clones. J. Exp. Med. 1989; 170:2081.
    37. Hsu, D. H., K. W. Moore, and H. Spits. Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity. Int. Immunol. 1992; 4:563.
    38. Strobel, S. Oral tolerance, systemic immunoregulation, and autoimmunity. Ann. N Y Acad. Sci. 2002; 958:47.
    39. Barnes, P. J. IL-10: a key regulator of allergic disease. Clin. Exp. Allergy 2001; 31:667.
    40. Francis, J. N., S. J. Till, and S. R. Durham. Induction of IL-10+CD4+CD25+ T-cells by grass pollen immunotherapy. J. Allergy Clin. Immunol. 2003; 111:1255.
    41. Morafo, V., K. Srivastava, C. K. Huang, G. Kleiner, S. Y. Lee, Sampson H A, and Li X. M. Genetic susceptibility to food allergy is linked to differential TH2-TH1 responses in C3H/HeJ and BALB/c mice. J. Allergy Clin. Immunol. 2003; 111:1122.
    42. Saarinen, K. M., O. Vaarala, P. Klemetti, and E. Savilahti. Transforming growth factor-beta1 in mothers' colostrum and immune responses to cows' milk proteins in infants with cows' milk allergy. J. Allergy Clin. Immunol. 1999; 104:1093.


Example 13
In Vivo Experiments Demonstrating the Long-Term Benefits of Desensitization
This Example refers to various patents, publications, books, articles, and other references that are listed under Section 13.5. The contents of all of these items are hereby incorporated by reference in their entirety.
13.1 Introduction
The experiments that are described in this Example build on the desensitization results of Example 12 by investigating the long-term immunomodulatory effects of rectally administered HKE-MPE123. After several weeks of sensitization, peanut allergic C3H/HeJ mice received rectal administrations of 0.9 (low dose), 9 (medium dose) or 90 (high dose) μg HKE-MP123, HKE-containing vector (HKE-V) alone, or vehicle alone (sham) weekly for 3 weeks. Mice were challenged 2 weeks later (week 14). A second and third challenge were performed at 4-week intervals (weeks 18 and 22). Following the first challenge, all three HKE-MP123 and HKE-V-treated groups exhibited reduced symptom scores (p<0.01, 0.01, 0.05 and 0.05, respectively) as compared with the sham-treated group. Only the medium and high dose HKE-MP123-treated mice remained protected at week 22. IgE levels were significantly lower in all HKE-MP123 treated groups (p<0.001), being most reduced in the high dose HKE-MP123 treated group at the time of each challenge. IL-4, IL-13, IL-5, and IL-10 production by splenocytes of high dose HKE-MP123-treated mice were significantly decreased (p<0.01, 0.001, 0.001 and 0.001, respectively); and both IFN-γ and TGF-β production were significantly increased (p<0.001 and 0.01, respectively) as compared with sham-treated mice at the time of the last challenge. These results indicate that treatment with rectally administered HKE-MP123 can induce long-term “down-regulation” of peanut hypersensitivity, which may be secondary to decreased antigen-specific Th2 and increased Th1 and T regulatory cytokine production.
13.2 Materials and Methods
All materials and methods that are not described under this Example were obtained, prepared or performed as described in Example 12, Sections 12.2-12.3.
Intragastric Antigen-Sensitization, Challenge, and HKE-MP123 Treatment
Mice were sensitized with peanut and CT as described in Example 12, Section 12.3 (see also Ref. 11). As depicted in FIG. 30, treatment began at week 10 after the initial peanut sensitization. Six groups (twelve mice per group) were involved. Mice were treated with HKE-MP123 (G2=0.9 μg low dose; G3=9 μg medium dose; G4=90 μg high dose) or HKE-V (G5). Sham (saline)-treated (G1) and naïve (G8) mice were included as controls. Treatments were administered in 90 μl of methylcellulose as vehicle rectally 3 times at weekly intervals. Mice were challenged intragastrically 2, 6 and 10 weeks post-therapy (weeks 14, 18, and 22 post-initial sensitization). Following each challenge, 4 mice were sacrificed to collect samples for immunologic studies.
13.3 Results
HKE-MP123 Confers Long Lasting Protection Against Peanut-Induced Anaphylaxis Following Oral Peanut Challenge
To determine whether HKE-MP123 can provide a long lasting effect on peanut allergy, peanut-sensitized mice were treated with 3 different weekly doses of rectally administered HKE-MP123 in a methylcellulose carrier. Mice were then challenged ig with peanut 2 weeks later (week 14 after the initial sensitization) and again 4 and 8 weeks later (weeks 18 and 22 respectively after the initial sensitization). Anaphylactic symptom scores were evaluated 30 minutes after challenge. Following the first challenge, all three HKE-MP123-treated groups exhibited significantly lower anaphylactic symptom scores compared to the sham-treated group (low, medium and high dose HKE-MP123-treated groups vs. sham: p<0.01, 0.01 and 0.01, respectively, FIG. 31A). No dose response difference was observed among the HKE-MP123-treated groups at the time of the first challenge. HKE-V also significantly reduced symptom scores compared to the sham-treated group (p<0.05), although the symptom scores tended to be greater in this group compared to the HKE-MP123-treated groups. Following the second challenge (week 18), anaphylactic symptom scores were reduced significantly only in the medium and high dose HKE-MP123-treated groups (p<0.05 and p<0.01, respectively, FIG. 31B). Similarly, at the third challenge (week 22), only mice receiving the medium and high doses of HKE-MP123 were protected from anaphylactic reactions (p<0.01, FIG. 31C). These results demonstrate that higher doses of HKE-MP123 in a methylcellulose carrier resulted in persistent protection lasting at least 10 weeks.
HKE-MP123 has a Long Lasting Inhibitory Effect on Peanut-Induced Histamine Release
Since histamine is associated with the anaphylactic reactions, we also measured plasma histamine levels 30 minutes following each peanut challenge. We found that following the first challenge at week 14, plasma histamine levels were significantly reduced in all three HKE-MP123-treated groups as compared with the sham-treated group (p<0.01, FIG. 32A). Reduction of plasma histamine levels in the HKE-V-treated group failed to reach statistical significance. Following the second challenge, only the high dose HKE-MP123-treated group had significantly lower plasma histamine as compared with the sham-treated group (p<0.01, FIG. 32B). Following the third challenge, histamine levels were significantly lower in both the medium and high dose HKE-MP123-treated groups as compared with sham-treated group (p<0.01, FIG. 32C). Mice treated with low dose of HKE-MP123 and HKE-V did not show a significant reduction in plasma histamine levels as compared with the untreated group following the second and the third challenge (FIG. 32A, B, C). These results parallel the clinical findings in that HKE-MP123 (at medium and high doses) have a long lasting suppressive effect on histamine release, which lasted for at least 10 weeks
HKE-MP123 has a Long Lasting Effect on Peanut-Specific IgE and IgG2a Production
Peanut-specific IgE levels were monitored during sensitization/boosting, desensitization and following treatment. IgE levels increased markedly over the 8 week sensitization/boosting in each group of mice following peanut sensitization and were similar among the groups prior to treatment at week 10. Following treatment IgE levels were significantly reduced in all HKE-MP123-treated groups at the first, second and the third challenge (p<0.001, FIG. 33A) being lowest in the high dose treatment group. IgE levels were also reduced in HKE-V-treated group at the time of the third challenge (p<0.05), but were significantly greater than in the high dose HKE-MP123-treated group (p<0.05).
IgG2a levels were significantly increased in HKE-MP123 medium and high dose-treated groups at the first (p<0.01 and 0.001, respectively), the second (p<0.05 and 0.01, respectively) and the third challenges (p<0.05 and 0.001, respectively) as compared with the sham-treated group (FIG. 33B). IgG2a levels in the low dose HKE-MP123-treated group were also significantly greater than in the sham-treated group at the time of the first and second challenge, but not at the third challenge. IgG2a levels in the HKE-V-treated group were not significantly different than those of the sham-treated group at the time of the first and the second challenge.
These results indicate that HKE-MP123 suppresses IgE and increases IgG2a production. This effect lasted at least 10 weeks after discontinuing therapy, and the high doses of HKE-MP123 appeared to be the most effective.
HKE-MP123 Modulation of Th1, Th2 and T Regulatory Cytokines
To determine whether the long-lasting HKE-MP123-mediated protection against peanut allergy was associated with altered SPC cytokine profiles, we analyzed cytokine levels in SPC culture supernatants from each group of mice following the last challenge. IL-4 levels were significantly lower in the low, medium, and high dose HKE-MP123-treated and HKE-V-treated groups compared to the sham-treated group (p<0.01, 0.05, 0.01 and 0.05 respectively; FIG. 34A). IL-13 levels also were significantly decreased in the medium and high dose HKE-MP123 groups and the HKE-V group (p<0.01, FIG. 34B). However, significant reduction of IL-5 levels was only seen in the medium and high HKE-MP123-treated groups (p<0.001, FIG. 34C). IFN-γ levels were increased in all HKE-MP123-treated and HKE-V-treated groups (p<0.01, 0.001, 0.001 and 0.001, respectively; FIG. 34D).
As noted in Example 12, IL-10 is a classic Th2 cytokine believed to be involved in the induction of oral tolerance (see Ref 18) and the down-regulation of the allergic response (see Ref 19). TGF-β is also felt to be important in the development of oral tolerance to food allergens (see Ref 20). We found that IL-10 was reduced in all HKE-MP123-treated and HKE-V-treated groups as compared with the sham-treated group, being lowest in the HKE-MP123 high dose treated group (p<0.01, FIG. 34E). In contrast, TGF-β levels were significantly increased only in the HKE-MP123-treated groups in a dose dependent manner (p<0.05, 0.01 and 0.01; FIG. 34F). These results demonstrate that HKE-MP123 has a broad immunoregulatory effect on Th1, Th2 and T regulatory cytokines, which may contribute to its beneficial effect on peanut allergy.
13.4 Discussion
In the present Example, we have demonstrated that three rectal treatments with HKE-MP123 at medium (9 μg) or high doses (90 μg) provides peanut-allergic mice with significant protection from anaphylaxis for at least 10 weeks following the discontinuation of therapy. Low dose (0.9 μg) HKE-MP123, MP123 and HKE-V alone induced temporary protection, i.e., protection against the first challenge, but not subsequent challenges. These results demonstrate that the rectal administration of HKE producing engineered peanut proteins is efficacious for treating peanut allergy.
In addition to suppressing clinical symptoms, we found that HKE-MP123 produced long lasting suppression of histamine release following peanut challenge and a decrease in peanut-specific IgE levels. Peanut-specific IgE levels were also reduced in HKE-V-treated group, but were significantly greater than that seen in the high-dose HKE-MP123-treated group. This may have been due to the effect of CpG motifs in the plasmid vector. IgE levels were not significantly different in mice treated with the different doses of HKE-MP123 suggesting that the reduction in IgE is not solely responsible for the long lasting protection mediated by HKE-MP123. IgG2a levels were significantly increased for at least 10 weeks in the medium- and high-dose HKE-MP123-treated groups, and were associated with the long lasting protection in these two groups. IgG2a, a Th1 driven antibody (see Refs. 23-26) generally considered to be a “blocking antibody” (see Ref 27), was enhanced by HKE-MP123 treatment and may have been at least in part responsible for the long lasting beneficial effect of immunotherapy in this model.
Numerous studies have demonstrated that Th2 cytokines play a central role in the pathogenesis of allergic disorders, including food allergy. IL-4 and IL-13 promote B-cell switching to IgE production and mast cell activation, while IL-5 has been shown to have a potentially autocrine effect on mast cells, in addition to its recognized paracrine effects on eosinophils (see Refs. 28-29). IFN-γ, on the other hand, inhibits Th2 cell activation and mast cell/basophil mediator release upon re-exposure to antigen (see Refs. 30-31). Schade et al. recently demonstrated that T-cell clones generated from infants with cow milk allergy produced high levels of IL-4, IL-5 and IL-13, and low levels of IFN-γ, whereas T-cell clones produced from infants without cow milk allergy had high levels of IFN-γ and low levels of IL-4, IL-5, and IL-13 (see Ref 32). In addition, decreased IFN-γ was correlated with increased IgE levels in peanut allergic patients, and Th2 clones have been generated from patients with peanut allergy (see Refs. 33-34). Allergen-based immunotherapies are believed to reestablish immunologic tolerance to allergen by redirecting T-cell immune responses from a Th2- to Th1-type responses (see Ref 35). In the present study, we found that 10 weeks post-therapy, SPCs from mice treated with the higher doses of HKE-MP123 induced significant reductions of Th2 cytokines and increases in IFN-γ, suggesting a shift from Th2 responses to Th1 responses. The low-dose HKE-MP123-treated group and the HKE-V-treated group both showed induction of IFN-γ, and selective suppression of IL-4, and/or IL-13, but no effect on IL-5 production. These results suggest that higher doses of HKE-MP123 are more effective in regulating Th1 and Th2 responses, which may be associated with the long lasting therapeutic effect on peanut allergy.
While the counter-regulatory effect between Th1 and Th2 responses remains an important paradigm, an appreciation of the regulatory role of TGF-β and IL-10 has developed for both Th1-mediated autoimmune and Th2-mediated allergic responses (see Refs. 18-19, 36 and 38). Colostrum TGF-β concentrations were found to be lower in samples from mothers of infants with IgE mediated cow milk allergy than in samples from mothers of infants with non-IgE mediated cow milk allergy (see Ref 39). A recent study found that IL-10 was essential in parasite infection-mediated protection against peanut allergy in a murine model (see Ref 40). However, any relationship between TGF-β, IL-10 and allergen immunotherapy-mediated regeneration of oral tolerance to food antigen has not been demonstrated. In this Example, we found that TGF-β levels were significantly increased in all HKE-MP123-treated groups, but not the HKE-V-treated group, and appeared to be dose dependent. These results suggest that the induction of TGF-β might also be important for the long lasting therapeutic benefit of HKE-MP123 on peanut allergy. In addition, we found that IL-10 levels were reduced in all three HKE-MP123- and HKE-V-treated groups as compared with the sham-treated group, being lowest in the high dose HKE-MP123-treated group. These results suggest that IL-10 may play a less significant role in the HKE-MP123-mediated protective effect on peanut allergy. We and others recently found that increased IL-10 production appeared to be associated with induction of peanut allergy (see Refs. 40-41). In a study utilizing the co-administration of heat-killed L. monocytogenes and OVA in OVA sensitized mice, the suppression of IL-4 and increase in IFN-γ production was associated with a reduction of IL-10 (see Ref 10). The key cytokine(s) and cellular mechanisms responsible for the long-lasting protection against peanut anaphylaxis induced by HKE-MP123 in this study are unknown.
The HKE-V treatment also induced statistically significant protection (although less than HKE-MP123) at the first challenge, which may be due to vector CpGs within the E. coli resulting in switching the Th2 response to a Th1 response. However, the HKE-V effect on peanut allergy is unlikely attributable to the vector alone because mock DNA (plasmid DNA alone) had essentially no effect on allergy in a previous study (see Ref. 8).
In conclusion, this Example demonstrates that the rectal administration of high dose HKE-MP123 has a potent and persistent, therapeutic effect on peanut allergy in this model of peanut hypersensitivity. Protection lasted for at least 10 weeks, and was accompanied by persistent reduction of peanut-specific IgE and plasma histamine levels following challenges. The precise mechanisms associated with this long-lasting protection are not fully understood, but the results suggest that the protective effect is likely related to the down-regulation of Th2 cytokines, perhaps resulting from up-regulation of IFN-γ and TGF-β.
13.5 References


    1. Sampson H A. Food allergy. Part 1: immunopathogenesis and clinical disorders. J. Allergy Clin. Immunol. 1999; 103:717-728.
    2. Yocum M W, Butterfield J H, Klein J S, Volcheck G W, Schroeder D R, Silverstein M D. Epidemiology of anaphylaxis in Olmsted County: A population-based study. J. Allergy Clin. Immunol. 1999; 104:452-456.
    3. Sampson H A, Mendelson L, Rosen J P. Fatal and near-fatal anaphylactic reactions to food in children and adolescents. N. Engl. J. Med. 1992; 327:380-384.
    4. Bock S A, Munoz-Furlong A, Sampson H A. Fatalities due to anaphylactic reactions to foods. J. Allergy Clin. Immunol. 2001; 107:191-193.
    5. Sicherer S H, Munoz-Furlong A, Burks A W, Sampson H A. Prevalence of peanut and tree nut allergy in the US determined by a random digit dial telephone survey. J. Allergy Clin. Immunol. 1999; 103:559-562.
    6. Bock S A. The natural history of food sensitivity. J Allergy Clin Immunol 1982; 69:173-177.
    7. Oppenheimer J J, Nelson H S, Bock S A, Christensen F, Leung D Y. Treatment of peanut allergy with rush immunotherapy. J. Allergy Clin. Immunol. 1992; 90:256-262.
    8. Li X, Huang C K, Schofield B H, Burks A W, Bannon G A, Kim K H, Huang S K, Sampson H A. Strain-dependent induction of allergic sensitization caused by peanut allergen DNA immunization in mice. J. Immunol. 1999; 162:3045-3052.
    9. Huang C K, Schofield B H, Burks A W, Bannon G A, Huang S K, Sampson H, Li X M. Strain-dependent protection from allergic reactions by peanut allergen plasmid DNA immunization in mice. J. Allergy Clin. Immunol. 1999; 103:S238 (Abstract)
    10. Yeung V P, Gieni R S, Umetsu D T, DeKruyff R H. Heat-killed Listeria monocytogenes as an adjuvant converts established murine Th2-dominated immune responses into Th1-dominated responses. J Immunol 1998; 161:4146-4152.
    11. Li X M, Srivastava K, Huleatt J W, Bottomly K, Burks A W, Sampson H A. Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model. J Immunol 2003; 170:3289-3295.
    12. Institute of Laboratory Animal Resources Commission of Life Sciences NRC. Guide for the Care and Use of Laboratory Animals. National Academy Press, 1996:
    13. Li X M, Serebrisky D, Lee S Y, Huang C K, Bardina L, Schofield B H, Stanley J S, Burks A W, Bannon G A, Sampson H A. A murine model of peanut anaphylaxis: T- and B-cell responses to a major peanut allergen mimic human responses. J. Allergy Clin. Immunol. 2000; 106:150-158.
    14. Stanley J S, King N, Burks A W, Huang S K, Sampson H, Cockrell G, Helm R M, West C M, Bannon G A. Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2. Arch. Biochem. Biophys. 1997; 342:244-253.
    15. Burks A W, King N, Bannon G A. Modification of a major peanut allergen leads to loss of IgE binding. Int Arch Allergy Immunol 1999; 118:313-314.
    16. Li X M, Schofield B H, Huang C K, Kleiner G A, Sampson H A. A Murine Model of IgE Mediated Cow Milk Hypersensitivity. J. Allergy Clin. Immunol. 1999; 103:206-214.
    17. Lee S Y, Huang C K, Zhang T F, Schofield B H, Burks A W, Bannon G A, Sampson H A, Li X M. Oral Administration of IL-12 Suppresses Anaphylactic Reactions in a Murine Model of Peanut Hypersensitivity. Clin Immunol 2001; 101:220-228.
    18. Strobel S. Oral tolerance, systemic immunoregulation, and autoimmunity Ann N Y Acad Sci 2002; 958:47-58.
    19. Barnes P J. IL-10: a key regulator of allergic disease. Clin Exp Allergy 2001; 31:667-669.
    20. Husby S. Sensitization and tolerance. Curr Opin Allergy Clin Immunol 2001; 1:237-241.
    21. Leung D Y, Sampson H A, Yunginger J W, Burks A W J, Schneider L C, Wortel C H, Davis F M, Hyun J D, Shanahan W R J. Effect of anti-IgE therapy in patients with peanut allergy. N Engl J Med 2003; 348:986-993.
    22. Li X. M., Sampson H A. Novel approaches for the treatment of food allergy. Current Opinion in Allergy and Clinical Immunology 2002; 2:273-278.
    23. Snapper C M, Paul W E. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 1987; 236:944-947.
    24. Howard M, Paul W E. Regulation of B-cell growth and differentiation by soluble factors. Annu Rev. Immunol. 1983; 1:307-333.
    25. Mosmann T R, Coffman R L. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev. Immunol. 1989; 7:145-173.
    26. Beck L, Spiegelberg H L. The polyclonal and antigen-specific IgE and IgG subclass response of mice injected with ovalbumin in alum or complete Freund's adjuvant. Cell Immunol. 1989; 123:1-8.
    27. Kowalski M L, Jutel M. Mechanisms of specific immunotherapy of allergic diseases. Allergy 1998; 53:485-492.
    28. Lorentz A, Schwengberg S, Mierke C, Manns M P, Bischoff S C. Human intestinal mast cells produce IL-5 in vitro upon IgE receptor cross-linking and in vivo in the course of intestinal inflammatory disease. Eur J Immunol 1999; 29:1496-1503.
    29. Romagnani S. The role of lymphocytes in allergic disease. J. Allergy Clin. Immunol. 2000; 105:399-408.
    30. Bissonnette E Y, Befus A D. Inhibition of mast cell-mediated cytotoxicity by IFN-alpha/beta and -gamma. J Immunol 1990; 145:3385-3390.
    31. Pierkes M, Bellinghausen I, Hultsch T, Metz G, Knop J, Saloga J. Decreased release of histamine and sulfidoleukotrienes by human peripheral blood leukocytes after wasp venom immunotherapy is partially due to induction of IL-10 and IFN-gamma production of T-cells. J Allergy Clin Immunol 1999; 103:326-332.
    32. Schade R P, Ieperen-Van Dijk A G, Van Reijsen F C, Versluis C, Kimpen J L, Knol E F, C. A., Van Hoffen E. Differences in antigen-specific T-cell responses between infants with atopic dermatitis with and without cow's milk allergy: relevance of TH2 cytokines J. Allergy Clin. Immunol. 2000; 106:1155-1162.
    33. de Jong E C, Van Zijverden M, Spanhaak S, Koppelman S J, Pellegrom H, Penninks A H. Identification and partial characterization of multiple major allergens in peanut proteins. Clin. Exp. Allergy 1998; 28:743-751.
    34. Dorion B J, Burks A W, Harbeck R, Williams L W, Trumble A, Helm R M, Leung D Y. The production of interferon-gamma in response to a major peanut allergy, Ara h II correlates with serum levels of IgE anti-Ara h II. J. Allergy Clin. Immunol. 1994; 93:93-99.
    35. Durham S R, Till S J. Immunologic changes associated with allergen immunotherapy. J Allergy Clin Immunol 1998; 102:157-164.
    36. Donnet-Hughes A, Duc N, Serrant P, Vidal K, Schiffrin E J. Bioactive molecules in milk and their role in health and disease: the role of transforming growth factor-beta. Immunol Cell Biol 2000; 78:74-79.
    37. Zuany-Amorim C, Sawicka E, Manlius C, Le Moine A, Brunet L R, Kemeny D M, Bowen G, Rook G, Walker C. Suppression of airway eosinophilia by killed Mycobacterium vaccae-induced allergen-specific regulatory T-cells. Nat Med 2002; 8:625-629.
    38. Kalliomaki M, Ouwehand A, Arvilommi H, Kero P, Isolauri E. Transforming growth factor-beta in breast milk: a potential regulator of atopic disease at an early age. J Allergy Clin Immunol 1999; 104:1251-1257.
    39. Saarinen K M, Vaarala O, Klemetti P, Savilahti E. Transforming growth factor-beta1 in mothers' colostrum and immune responses to cows' milk proteins in infants with cows' milk allergy. J Allergy Clin Immunol 1999; 104:1093-1098.
    40. Bashir M E, Andersen P, Fuss I J, Shi H N, Nagler-Anderson C. An enteric helminth infection protects against an allergic response to dietary antigen. J Immunol 2002; 169:3284-3292.
    41. Morafo V, Srivastava K, Huang C K, Kleiner G, Lee S Y, Sampson H A, Li X. M. Genetic susceptibility to food allergy is linked to differential th2-th1 responses in C3H/HeJ and BALB/c mice. J. Allergy Clin. Immunol. 2003; 111:1122.


Example 14
Clinical Study of Rectally Delivered HKE-MP123 in Human Subjects
14.1 Introduction
Briefly, peanut-allergic subjects will receive 8 weekly rectal administrations of HKE-MP123 of increasing doses, followed by 3 bi-weekly administrations of the highest dose. Without limitation, the starting dose is currently estimated to be about 90 μg of encapsulated modified peanut protein (i.e., consisting of about 30 μg of each of the three modified peanut proteins within heat-killed E. coli), and will be doubled each week, if no adverse events occur (e.g., diarrhea, anaphylactic reactions, etc.) to a maximum dose of about 11,520 μg of modified peanut protein (i.e., about 3,840 μg of each of the three modified peanut proteins). Serum peanut-specific IgE levels and prick skin test (PST) titration responses will be measured prior to initiating desensitization and at weeks 4, 8, 12, and 14 of the study to assess the immunologic response to treatment over the course of the study. Blood may also be taken for basophil histamine release assays and optionally for T-cell activation assays. Finally, all subjects who undergo the full desensitization protocol will be challenged by progressive administration of an extract of whole peanut under controlled conditions at the hospital.
The following aspects of the clinical study are described in greater detail below: 1) study population, 2) study design, 3) study duration, 4) statistical plan, 5) rationale for starting dose and dose escalation scheme, 6) rationale for schedule and duration of administration, and 7) rationale for challenge.
14.2 Study Population
Approximately 12 male or female subjects between the ages of 18-55 years of age with peanut allergy will be enrolled in this study. Subjects will have a documented history of systemic responses to peanut exposure (e.g., including any of the following symptoms: urticaria and/or angioedema, lower respiratory symptoms, and hypotension), and a positive prick skin test and/or serum titer of peanut specific IgE greater than or equal to 5 kilounits of allergen (KUA).
Subjects will be excluded from the study if they 1) have suffered an acute illness within one week of the start of the study; 2) have a history of significant neurologic, hepatic, renal, endocrine, cardiovascular, gastrointestinal, pulmonary or metabolic disease; 3) show abnormal hepatic function (SGOT/SGPT and bilirubin >1.25× upper limit of normal); 4) show abnormal renal function (BUN and creatine >1.25× upper limit of normal); show abnormal bone marrow function (WBC, 4×103/mm3; platelets <100×103/mm3; hemoglobin <11 g/dl); 5) have a clinically significant abnormal electrocardiogram; 6) have used systemic steroids within 14 days of screening or during the trial; 7) have used aspirin within 3 days of the screening visit or during dosing visits; 8) have a history of alcohol or drug abuse; 9) are known to have hepatitis or HIV; 10) have participated in another experimental therapy study within 30 days prior to enrollment in this study; 11) have previously enrolled in this study; or 12) are pregnant or lactating.
14.3 Study Design
The study will be an open label, single center, safety and efficacy study of multiple rectal administrations of HKE-MP123 in subjects allergic to peanuts. The study will consist of two parts, the first part will assess the preliminary efficacy of HKE-MP123 to demonstrate desensitization two weeks post-treatment, and the second part will assess the duration of the desensitization post-treatment. The flow charts of FIGS. 35 and 36 outline these two parts.
In the first part of the proposed clinical trial for HKE-MP123, eligible subjects who have signed informed consent will be admitted to a medical center with experience in treating severe allergic reactions. A prick skin test (PST) titration will be conducted with standardized peanut extract to obtain a pre-treatment PST score. Subjects with a positive PST score will be eligible to continue in the study, and the PST score will serve as a pre-treatment value against which efficacy will be measured over the course of treatment and post-treatment. Peanut-specific basophil activation and serum peanut-specific IgE levels will also be measured pre-treatment, and will serve as a pre-treatment value against which efficacy will be measured over the course of treatment and post-treatment. T-cell activation assays may also be performed.
Subjects with a positive PST score will be administered one dose of HKE-MP123 rectally once every week for a total of 8 weeks. The current anticipated starting dose will be 90 μg of total modified peanut protein (i.e., 30 μg of each of the three modified peanut proteins), and will be doubled each week, if no adverse events occur, to a maximum dose of 11,520 μg of total modified peanut protein (i.e., 3,840 μg of each of the three modified peanut proteins). Subjects will then receive the maximum HKE-MP123 dose rectally once every two weeks for a total of 6 weeks.
Subjects will remain in the clinic each day of administration for 8 hours. Subjects will have vital signs monitored following each of the administrations and will be queried regarding any adverse events they experience, as well as concomitant medication use. Serum chemistries, including hepatic profiles and renal profiles, urinalyses, and complete blood counts (CBCs) will be monitored for all subjects prior to the initiation of therapy and at weeks 4, 8, 12, and 14. At weeks 4, 8, 12, and 14, prior to rectal administration of HKE-MP123, PST titrations will be conducted and peanut-induced basophil activation and serum peanut-specific IgE levels will be measured. Optionally T-cell activation will also be measured.
At week 28 of the study, after other assays have been conducted, subjects will be challenged with whole peanut. Optionally, in order to determine the long term effects of treatment, PST titration may be conducted and peanut-induced basophil activation and serum peanut-specific IgE levels may be remeasured at later dates (e.g., 3-12 or more weeks after the first challenge). Optionally T-cell activation will also be measured.
14.4 Study Duration
The study duration for each subject, from baseline evaluation to final visit, will be approximately 14 weeks. The screening evaluations will be conducted within 2 weeks prior to the baseline evaluations. As noted, the study may be optionally extended to determine the long term effects of treatment.
14.5 Statistical Plan
Descriptive statistics will be used to evaluate safety and efficacy outcomes of this study. Safety will be assessed based on adverse events, vital signs, serum chemistries, urinalyses, and hematology. Efficacy will be assessed based on 1) prick skin test (PST) titration values during the treatment period and following treatment, compared to values prior to initiation of treatment, 2) dose of peanut extract required to activate patient basophils in vitro, 3) serum peanut-specific IgE levels during the treatment period and following treatment, compared to values prior to initiation of treatment, and 4) food challenge with whole peanut following treatment. Optionally a T-cell activation assay may also be used.
14.6 Starting Dose and Escalation Scheme
The starting dose is based on the dosages that have been shown to be safe and produce desensitization efficacy in mice (see Examples 11-14). The escalation scheme, doubling each week, is based upon standard immunotherapy practice. The highest dose may exceed the normal therapeutic dose, but should provide evidence that the dose can be increased further, as is sometimes necessary in treating bee-sting anaphylaxis patients.
14.7 Rationale for Schedule and Duration of Administration
The once weekly schedule of administration is based upon a well-established immunotherapy paradigm for escalation to “maintenance” doses of immunotherapeutic extracts. The 8 week period for scale-up administration was selected based upon an extrapolation of the murine model data. The biweekly schedule of administration and the 6 week term of the maintenance period were selected in order to ensure that the immune system has a sufficient period of time, with continued but not relentless exposure, to respond to the treatment and become desensitized.
14.8 Rationale for Challenge
All subjects who undergo the full desensitization protocol will be challenged by progressive administration of an extract of whole peanut under controlled conditions at the hospital. This challenge will both provide information about the effectiveness of the proposed therapy, and will allow an investigation of whether measurable immunologic markers can be correlated with likely response to challenge with/exposure to peanut. Unfortunately, to date no immunologic tests have been identified that can reliably predict the likelihood that a particular individual will or will not react to exposure to a given amount of peanut antigen. The present study tracks three specific immunologic markers over time: PST titration to peanut extract, peanut-specific basophil activation, and serum-specific peanut IgE. Optionally antigen-specific T-cell responses may also be assayed.
OTHER EMBODIMENTS
Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and Examples be considered as exemplary only, with the true scope of the invention being indicated by the following Claims.







APPENDIX 1







WEED POLLENS












SYSTEMATIC AND

SEQUENCE
ACCESSION NO.


ALLERGEN SOURCE
ORIGINAL NAMES
MW (KD)
DATA1
OR REFERENCES










Asterales











Ambrosia
Amb a 1; antigen E
38
C
8, 20


artemisiifolia
Amb a 2; antigen K
38
C
8, 21


(short ragweed)
Amb a 3; Ra3
11
C
22



Amb a 5; Ra5
5
C
11, 23



Amb a 6; Ra6
10
C
24, 25



Amb a 7; Ra7
12
P
26


Ambrosia trifida
Amb t 5; Ra5G
4.4
C
9, 10, 27


(giant ragweed)


Artemisia vulgaris
Art v 1
27-29
C
28


(mugwort)
Art v 2
35
P
28a



Art v 3; lipid transfer
12
P
53



protein



Art v 4; profilin
14
C
29


Helianthus annuus
Hel a 1
34
—
29A


(sunflower)
Hel a 2; profilin
15.7
C
Y15210


Mercurialis annua
Mer a 1; profilin
14-15
C
Y13271







Carophyllales











Chemopodium
Che a 1
17
C
29B, AY049012


album (lamb's
Che a 2; profilin
14
C
AY082337


quarters, pigweed,
Che a 3; polcalcin
10
C
AY082338


white goosefoot)


Salsola kali
Sal k 1
43
P
29C


(Russian thistle)







Rosales











Humulus
Hum j 4w

C
AY335187


japonicus


(Japanese hop)


Parietaria judaica
Par j 1; lipid transfer
15
C
X77414



protein 1



Par j 2; lipid transfer

C
X95865



protein 2



Par j 3; profilin

C
Y15208


Parietaria
Par o 1; lipid transfer
15

29D


officinalis
protein





1P = Protein sequence, C = cDNA sequence













APPENDIX 2







GRASS POLLENS












SYSTEMATIC AND

SEQUENCE
ACCESSION NO.


ALLERGEN SOURCE
ORIGINAL NAMES
MW (KD)
DATA
OR REFERENCES










Poales











Cynodon dactylon
Cyn d 1
32
C
30, S83343


(Bermuda grass)
Cyn d 7

C
31, X91256



Cyn d 12; profilin
14
C
31A, Y08390



Cyn d 15
 9
C
AF517686



Cyn d 22w; enolase


Pending



Cyn d 23; Cyn d 14
 9
C
AF517685



Cyn d 24;
21
P
Pending



pathogenesis-related



protein


Dactylis
Dac g 1; AgDg1
32
P
32


glomerata
Dac g 2
11
C
33, S45354


(orchard grass)
Dac g 3

C
33A, U25343



Dac g 5
31
P
34


Festuca pratensis
Fes p 4w
60


(meadow fescue)


Holcus lanatus
Hol l 1

C
Z27084


(velvet grass)


Lolium perenne
Lol p 1; group I
27
C
35, 36


(rye grass)
Lol p 2; group II
11
C
37, 37A, X73363



Lol p 3; group III
11
C
38



Lol p 5; Lol p IX,
31/35
C
34, 39



Lol p Ib



Lol p 11; trypsin
16

39A



inhibitor


Phalaris aquatica
Pha a 1

C
40, S80654


(canary grass)


Phleum pratense
Phl p 1
27
C
X78813


(timothy)
Phl p 2

C
41, X75925



Phl p 4

P
41A



Phl p 5; Ag25
32
C
42



Phl p 6

C
43, Z27082



Phl p 11; trypsin
20
C
43A, AF5211563



inhibitor



Phl p 12; profilin

C
44, X77583



Phl p 13;
55-60
C
AJ238848



polygalacturonase


Poa pratensis
Poa p 1; group I
33
P
46


(Kentucky blue
Poa p 5
31/34
C
34, 47


grass)


Sorghum
Sor h 1

C
48


halepense


(Johnson grass)
















APPENDIX 3







TREE POLLENS












SYSTEMATIC AND

SEQUENCE
ACCESSION NO.


ALLERGEN SOURCE
ORIGINAL NAMES
MW (KD)
DATA
OR REFERENCES










Arecales











Phoenix dactylifera
Pho d 2
14.3
C
Asturias (p.c.)


(date palm)







Fagales











Alnus glutinosa
Aln g 1
17
C
S50892


(alder)


Betula verrucosa
Bet v 1
17
C
X15877


(birch)
Bet v 2; profilin
15
C
M65179



Bet v 3

C
X79267



Bet v 4
8
C
X87153, S54819



Bet v 6; isoflavone
33.5
C
AF135127



reductase



Bet v 7; cyclophilin
18
P
P81531


Carpinus betulus
Car b 1
17
C
X66932


(hornbeam)


Castanea sativa
Cas s 1; Bet v 1
22
P
52


(chestnut)
homologue


(also a food)
Cas s 5; chitinase
9.7
P
53



Cas s 8; lipid transfer



protein


Corylus avellana
Cor a 1
17
C
X70999


(hazel)
Cor a 2; profilin
14
C


(also a food)
Cor a 8; lipid transfer
9
C



protein



Cor a 9; 11S
40
C
Beyer (p.c.)



globulin-like protein



Cor a 10 luminal
70
C
AJ295617



binding protein



Cor a 11; 7S vicilin-
48
C
AF441864



like protein


Quercus alba
Que a 1
17
P
54


(white oak)







Lamiales


Oleaceae











Fraxinus excelsior
Fra e 1
20
P
58A


(ash)


Ligustrum vulgare
Lig v 1
20
P
58A


(privet)


Olea europea
Ole e 1
16
C
59, 60


(olive)
Ole e 2; profilin
15-18
C
60A



Ole e 3
9.2

60B



Ole e 4
32
P
P80741



Ole e 5; superoxide
16
P
P80740



dismutase



Ole e 6
10
C
60C, U86342



Ole e 7

P
60D, P81430



Ole e 8; Ca2+-binding
21
C
60E, AF078679



protein



Ole e 9; beta-1,3-
46
C
AF249675



glucanase


Syringa vulgaris
Syr v 1
20
P
58A


(lilac)







Plantaginaceae











Plantago lanceolata
Pla l 1
18
P
P842242


(English plantain)







Pinales











Cryptomeria
Cry j 1
41-45
C
55, 56


japonica (sugi)
Cry j 2

C
57, D29772


Cupressus
Cup a 1
43
C
A1243570


arizonica (cypress)


Cupressus
Cup s 1
43
C
AF257491


sempervirens
Cup s 3w
34
C
Pending


(common cypress)


Juniperus ashei
Jun a 1
43
P
P81294


(mountain cedar)
Jun a 2

C
57A, AJ404653



Jun a 3
30
P
57B, P81295


Juniperus
Jun o 4;
29
C
57C, AF031471


oxycedrus
calmodulin-like


(prickly juniper)


Juniperus
Jun s 1
50
P
58


sabinoides


(mountain cedar)


Juniperus
Jun v 1
43
P
58B, P81825


virginiana


(eastern red cedar)







Platanaceae











Platanus acerifolia
Pla a 1
18
P
P82817


(London plane
Pla a 2
43
P
P82967


tree)
Pla a 3; lipid transfer
10
P
Iris (p.c.)



protein
















APPENDIX 4







MITE ALLERGENS












SYSTEMATIC AND

SEQUENCE
ACCESSION NO.


ALLERGEN SOURCE
ORIGINAL NAMES
MW (KD)
DATA
OR REFERENCES





Acarus siro
Aca s 13; fatty
 14*
C
AJ006774


(mite)
acid-binding protein





Blomia tropicalis
Blo t 1; cysteine
39
C
AF277840


(mite)
protease






Blo t 3; trypsin
 24*
C
Cheong (p.c.)



Blo t 4; alpha amylase
56
C
Cheong (p.c.)



Blo t 5

C
U59102



Blo t 6; chymotrypsin
25
C
Cheong (p.c.)



Blo t 10; tropomyosin
33
C
61



Blo t 11; paramyosin
110 
C
61A, AF525465



Blo t 12; Bt11a

C
U27479



Blo t 13; Bt6 fatty

C
U58106



acid-binding protein






Blo t 11; anti-microbial
  7.2
C
Cheong (p.c.)



protein





Dermatophagoides
Der f 1; cysteine
25
C
69


farinae (American
protease





house dust mite)
Der f 2
14
C
70, 70A



Der f 3; trypsin
30
C
63



Der f 7
24-31
C
71, SW: Q26456



Der f 10; tropomyosin

C
72



Der f 11; paramyosin
98
C
72A



Der f 14; Mag3,

C
D17686



apolipophorin






Der f 15; 98k chitinase
98
C
AF178772



Der f 16; gelsolin/villin
53
C
71A



Der f 17; Ca2+-binding
53
C
71A



EF protein






Der f 18w; 60k chitinase
60
C
Weber (p.c.)


Dermatophagoides
Der m 1; cysteine
25
P
68


microceras (mite)
protease





Dermatophagoides
Der p 1; antigen P1,
25
C
62


pteronyssinus
cysteine protease





(mite)
Der p 2
14
C
62A-C



Der p 3; trypsin
28/30
C
63



Der p 4; amylase
60
P
64



Der p 5
14
C
65



Der p 6; chymotrypsin
25
P
66



Der p 7
22/28
C
67



Der p 8; glutathione

C
67A



transferase






Der p 9; collagenolytic

P
67B



serine protein






Der p 10; tropomyosin
36
C
Y14906



Der p 14; apolipophorin

C
Epton (p.c.)



like protein





Europglyphus
Eur m 2

C
AF047613


maynei (mite)
Eur m 14; apolipophorin
177 
C
AF149827


Glycyphagus
Gly d 2

C
72B


domesticus






(storage mite)






Lepidoglyphus
Lep d 2
15
C
73, 74, 74A


destructor
Lep d 5

C
75, AJ250278


(storage mite)
Lep d 7

C
75, AJ271058



Lep d 10; tropomyosin

C
75A, AJ250096



Lep d 13

C
75, AJ250279


Tyrophagus
Tyr p 2

C
75B, Y12690


putrescentiae






(storage mite)
















APPENDIX 5







ANIMAL ALLERGENS











ALLERGEN
SYSTEMATIC AND

SEQUENCE
ACCESSION NO.


SOURCE
ORIGINAL NAMES
MW (KD)
DATA
OR REFERENCES














Bos domesticus
Bos d 2; Ag3, lipocalin
20
C
76, L42867


(domestic cattle)
Bos d 3; Ca2+-binidng
11
C
L39834


(also a food)
S100 homologue






Bos d 4;
14.2
C
M18780



alpha-lactalbumin






Bos d 5;
18.3
C
X14712



beta-lactoglobulin






Bos d 6; serum albumin
67
C
M73993



Bos d 7; immunoglobulin
160

77



Bos d 8; caseins
20-30

77


Canis familiaris
Can f 1
25
C
78, 79


(dog)
Can f 2
27
C
78, 79



Can f 3; albumin

C
S72946



Can f 4
18
P
A59491


Equus caballus
Equ c 1; lipocalin
25
C
U70823


(domestic horse)
Equ c 2; lipocalin
18.5
P
79A, 79B



Equ c 3; Ag3-albumin
67
C
79C, X74045



Equ c 4
17
P
79D



Equ c 5; AgX
17
P
Goutran Botros






(p.c.)


Felis domesticus
Fel d 1; cat-1
38
C
15


(cat saliva)
Fel d 2; albumin

C
79E, X84842



Fel d 3; cystatin
11
C
79F, AF238996



Fel d 4; lipocalin
22
C
AY497902



Fel d 5w; IgA
400

Adedoyin (p.c.)



Fel d 6w; IgM
800-1000

Adedoyin (p.c.)



Fel d 7w; IgG
150

Adedoyin (p.c.)


Cavia porcellus
Cav p 1; lipocalin
20
P
80, SW: P83507


(guinea pig)
homologue






Cav p 2
17
P
SW: P83508


Mus musculus
Mus m 1; MUP
19
C
81, 81A


(mouse urine)






Rattus
Rat n 1
17
C
82, 83


norvegius






(rat urine)
















APPENDIX 6







FUNGI ALLERGENS











ALLERGEN
SYSTEMATIC AND

SEQUENCE
ACCESSION NO.


SOURCE
ORIGINAL NAMES
MW (KD)
DATA
OR REFERENCES










Ascomycota


Dothidiales











Alternaria
Alt a 1
28
C
U82633


alternata
Alt a 2
25
C
83A, U62442



Alt a 3; heat shock
70
C
U87807, U87808



protein






Alt a 4; protein
57
C
X84217



disulfidisomerase






Alt a 6; acidic
11
C
X78222, U87806



ribosomal protein P2






Alt a 7; YCP4 protein
22
C
X78225



Alt a 10; aldehyde
53
C
X78227, P42041



dehydrogenase






Alt a 11; enolase
45
C
U82437



Alt a 12; acidic
11
C
X84216



ribosomal protein P1





Cladosporium
Cla h 1
13

83B, 83C


herbarum
Cla h 2
23

83B, 83C



Cla h 3; aldehyde
53
C
X78228



dehydrogenase






Cla h 4; acidic
11
C
X78223



ribosomal protein P2






Cla h 5; YCP4 protein
22
C
X78224



Cla h 6; enolase
46
C
X78226



Cla h 12; acidic
11
C
X85180



ribosomal protein P1










Eurotiales











Aspergillus flavus
Asp fl 13; alkaline
34

84



serine proteinase





Aspergillus
Asp f 1
18
C
M83781, S39330


fumigatus
Asp f 2
37
C
U56938



Asp f 3; peroxisomal
19
C
U20722



protein






Asp f 4
30
C
AJ001732



Asp f 5;
42
C
Z30424



metalloprotease






Asp f 6; Mn
  26.5
C
U53561



superoxide dismutase






Asp f 7
12
C
AJ223315



Asp f 8; ribosomal
11
C
AJ224333



protein P2






Asp f 9
34
C
AJ223327



Asp f 10; aspartic
34
C
X85092



protease






Asp f 11;
24

84A



peptidyl-prolyl isom






Asp f 12; heat shock
90
C
85



protein P90






Asp f 13; alkaline
34

84B



serine proteinase






Asp f 15
16
C
AJ002026



Asp f 16
43
C
g3643813



Asp f 17

C
AJ224865



Asp f 18; vacuolar
34

84C



serine proteinase






Asp f 22w; enolase
46
C
AF284645



Asp f 23; L3 ribosomal
44
C
85A, AF464911



protein





Aspergillus niger
Asp n 14;
105 
C
AF108944



beta-xylosidase






Asp n 18; vacuolar
34
C
84B



serine proteinase






Asp n ?
85
C
Z84377


Aspergillus
Asp o 13; alkaline
34
C
X17561


oryzae
serine proteinase






Asp o 21;
53
C
D00434, M33218



TAKA-amylase A





Penicillium
Pen b 13; alkaline
33

86A


brevicompactum
serine proteinase





Penicillium
Pen ch 13; alkaline
34

87


chrysogenum
serine proteinase






Pen ch 18; vacuolar
32

87



serine protease






Pen ch 20; N-acetyl
68

87A



glucosaminidase





Penicillium
Pen c 3; peroxisomal
18

86B


citrinum
membrane protein






Pen c 13; alkaline
33

86A



serine proteinase






Pen c 19; heat shock
70
C
U64207



protein P70






Pen c 22w; enolase
46
C
AF254643



Pen c 24; elongation

C
AY363911



factor 1 beta





Penicillium
Pen o 18; vacuolar
34

87B


oxalicum
serine proteinase










Hypocreales











Fusarium
Fus c 1; ribosomal
 11*
C
AY077706


culmorum
protein P2






Fus c 2; thioredoxin-
 13*
C
AY077707



like protein










Onygenales











Trichophyton
Tri r 2

C
88


rubrum
Tri r 4; serine protease

C
88


Trichophyton
Tri t 1
30
P
88A


tonsurans
Tri t 4; serine protease
83
C
88







Saccharomycetales











Candida albicans
Cand a 1
40
C
89



Cand a 3; peroxisomal
29
C
AY136739



protein





Candida boidinii
Cand b 2
20
C
J04984, J04985







Basidiomycota


Hymenomycetes











Psilocybe
Psi c 1





cubensis
Psi c 2; cyclophilin
16

89A


Coprinus comatus
Cop c 1; leucine zipper
11
C
AJ132235


(shaggy cap)
protein






Cop c 2


AJ242791



Cop c 3


AJ242792



Cop c 5


AJ242793



Cop c 7


AJ242794







Urediniomycetes











Rhodotorula
Rho m 1; enolase
47
C
89B


mucilaginosa











Ustilaginomycetes











Malassezia furfur
Mala f 2; MF1
21
C
90, AB011804



peroxisomal






membrane protein






Mala f 3; MF2
20
C
90, AB011805



peroxisomal






membrane protein






Mala f 4;
35
C
90A, AF084828



mitochondrial malate






dehydrogenase





Malassezia
Mala s 1

C
91, X96486


sympodialis
Mala s 5
 18*
C
AJ011955



Mala s 6
 17*
C
AJ011956



Mala s 7

C
91A, AJ011957



Mala s 8
 19*
C
91A, AJ011958



Mala s 9
 37*
C
91A, AJ011959



Mala s 10; heat shock
86
C
AJ428052



protein 79






Mala s 11; Mn
23
C
AJ548421



superoxide dismutase










Deuteromycotina


Tuberculariales











Epicoccum
Epi p 1; serine
30
P
91B; SW: P83340


purpurascens
protease
















APPENDIX 7







INSECT ALLERGENS











ALLERGEN
SYSTEMATIC AND ORIGINAL
MW
SEQUENCE
ACCESSION NO.


SOURCE
NAMES
(KD)
DATA
OR REFERENCES





Aedes aegyptii
Aed a 1; Apyrase
68
C
L12389


(mosquito)
Aed a 2;
37
C
M33157


Apis mellifera
Api m 1; phospholipase A2
16
C
92


(honey bee)
Api m 2; hyaluronidase
44
C
93



Api m 4; melittin
 3
C
94



Api m 6
7-8
P
Kettner (p.c.)



Api m7; CUB serine
39
C
AY127579



protease





Bombus
Bom p 1; phospholipase
16
P
95


pennsylvanicus
Bom p 4; protease

P
95


(bumble bee)






Blattella
Bla g 1; Bd90k

C



germanica
Bla g 2; aspartic protease
36
C
96


(German
Bla g 4; calycin
21
C
97


cockroach)
Bla g 5; glutathione transf.
22
C
98



Bla g 6; troponin C
27
C
98


Periplaneta
Per a 1; Cr-PII

C



americana
Per a 3; Cr-PI
72-78
C
98A


(American
Per a 7; tropomyosin
37
C
Y14854


cockroach)






Chironomus
Chi k 10; tropomyosin
  32.5*
C
AJ012184


kiiensis (midge)






Chironomus
Chi t 1-9; hemoglobin
16
C
99


thummi (midge)
Chi t 1.01; component III
16
C
P02229



Chi t 1.02; component IV
16
C
P02230



Chi t 2.0101; component I
16
C
P02221



Chi t 2.0102; component IA
16
C
P02221



Chi t 3; component II-beta
16
C
P02222



Chi t 4; component IIIA
16
C
P02231



Chi t 5; component VI
16
C
P02224



Chi t 6.01; component VIIA
16
C
P02226



Chi t 6.02; component IX
16
C
P02223



Chi t 7; component VIIB
16
C
P02225



Chi t 8; component VIII
16
C
P02227



Chi t 9; component X
16
C
P02228


Ctenocephalides
Cte f 1





felis felis
Cte f 2; M1b
27
C
AF231352


(cat flea)






Thaumetopoea
Tha p 1
15
P
99A,


pityocampa



PIR: A59396


(pine process.






moth)






Lepisma
Lep s 1; tropomyosin
36
C
AJ309202


saccharina






(silverfish)






Dolichovespula
Dol m 1; phospholipase A1
35
C
100


maculata
Dol m 2; hyaluronidase
44
C
101


(white face
Dol m 5; antigen 5
23
C
102, 103


hornet)






Dolichovespula
Dol a 5; antigen 5
23
C
104


arenaria






(yellow hornet)






Polistes
Pol a 1; phospholipase A1
35
P
105


annularies
Pol a 2; hyaluronidase
44
P
105


(wasp)
Pol a 5; antigen 5
23
C
104


Polistes
Pol d 1


Hoffman (p.c.)


dominulus
Pol d 4; serine protease
32-34
C
Hoffman (p.c.)


(Mediterranean
Pol d 5


P81656


paper wasp)






Polistes
Pol e 1; phospholipase A1
34
P
107


exclamans
Pol e 5; antigen 5
23
C
104


(wasp)






Polistes fuscatus
Pol f 5; antigen 5
23
C
106


(wasp)






Polistes metricus
Pol m 5; antigen 5
23
P
106


(wasp)






Vespa crabo
Vesp c 1; phospholipase
34
P
107


(European hornet)
Vesp c 5; antigen 5
23
C
106


Vespa mandarina
Vesp m 1


Hoffman (p.c.)


(giant asian
Vesp m 5


P81657


hornet)






Vespula
Ves f 5; antigen 5
23
C
106


flavopilosa






(yellowjacket)






Vespula
Ves g 5; antigen 5
23
C
106


germanica






(yellowjacket)






Vespula
Ves m 1; phospholipase A1
  33.5
C
108


maculifrons
Ves m 2; hyaluronidase
44
P
109


(yellowjacket)
Ves m 5; antigen 5
23
C
104


Vespula
Ves p 5; antigen 5
23
C
106


pennsylvanica






(yellowjacket)






Vespula
Ves s 5; antigen 5
23
C
106


squamosa






(yellowjacket)






Vespula vidua
Ves vi 5
23
C
106


(wasp)






Vespula vulgaris
Ves v 1; phopholipase A1
35
C
105A


(yellowjacket)
Ves v 2; hyaluronidase
44
P
105A



Ves v 5; antigen 5
23
C
104


Myrmecia
Myr p 1

C
X70256


pilosula
Myr p 2

C
S81785


(Australian






jumper ant)






Solenopsis
Sol g 2


Hoffman (p.c.)


geminata
Sol g 4


Hoffman (p.c.)


(tropical fire ant)






Solenopsis invicta
Sol i 2
13
C
110, 111


(fire ant)
Sol i 3
24
C
110



Sol i 4
13
C
110


Solenopsis
Sol s 2


Hoffman (p.c.)


saevissima






(brazilian fire ant)






Triatoma
Tria p 1; procalin
20
C
111A,


procracta



AF179004


(Californian






kissing bug)
















APPENDIX 8







FOOD ALLERGENS











ALLERGEN
SYSTEMATIC AND

SEQUENCE
ACCESSION NO. OR


SOURCE
ORIGINAL NAMES
MW (KD)
DATA
REFERENCES





Gadus callarias
Gad c 1; allergen M
12
C
112, 113


(cod)






Salmo salar
Sal s 1; parvalbumin
12
C
X97824


(Atlantic salmon)



X97825


Bos domesticus
Bos d 4; alpha-
  14.2
C
M18780


(domestic cattle)
lactalbumin





(milk)
Bos d 5; beta-
  18.3
C
X14712


(see also animals)
lactoglobulin






Bos d 6; serum albumin
67
C
M73993



Bos d 7; immunoglobulin
160 

77



Bos d 8; caseins
20-30

77


Gallus
Gal d 1; ovomucoid
28
C
114, 115


domesticus
Gal d 2; ovalbumin
44
C
114, 115


(chicken)
Gal d 3; Ag22,
78
C
114, 115



conalbumin






Gal d 4; lysozyme
14
C
114, 115



Gal d 5; serum albumin
69
C
X60688


Metapenaeus
Met e 1; tropomyosin

C
U08008


ensis






(shrimp)






Penaeus aztecus
Pen a 1; tropomyosin
36
P
116


(shrimp)






Penaeus indicus
Pen i 1; tropomyosin
34
C
116A


(shrimp)






Penaeus
Pen m 1; tropomyosin
38
C



monodon
Pen m 2; arginine kinase
40
C
117, AF479772


(black tiger






shrimp)






Todarodes
Tod p 1; tropomyosin
38
P
117A


pacificus (squid)






Helix aspersa
Hel as 1; tropomyosin
36
C
117B, Y14855


(brown garden






snail)






Haliotis Midae
Hal m 1
49

117C


(abalone)






Brassica juncea
Bra j 1; 2S albumin
14
C
118


(oriental mustard)






Brassica napus
Bra n 1; 2S albumin
15
P
118A, P80208


(rapeseed)






Brassica rapa
Bra r 2; prohevein-like
25

P81729


(turnip)
protein





Hordeum vulgare
Hor v 15; BMAI-1
15
C
119


(barley)
Hor v 16; alpha-amylase






Hor v 17; beta-amylase






Hor v 21; gamma-3
34
C
119A, SW: P80198



hordein





Secale cereale
Sec c 20; secalin


PIR: S70327


(rye)






Triticum
Tri a 18; agglutinin





aestivum
Tri a 19; omega-5 gliadin
65
P
PIR: A59156


(wheat)






Zea mays
Zea m 14; lipid transfer
 9
P
P19656


(maize, corn)
protein





Oryza sativa
Ory s 1

C
119B, U31771


(rice)






Apium
Api g 1; Bet v 1
 16*
C
Z48967


graveolens
homologue





(celery)
Api g 4; profilin


AF129423



Api g 5
55/58
P
P81943


Daucus carota
Dau c 1; Bet v 1
16
C
117D


(carrot)
homologue






Dau c 4; profilin

C
AF456482


Malus domestica
Mal d 1; Bet v 1

C
X83672


(apple)
homologue






Mal d 2; thaumatin

C
AJ243427



homologue






Mal d 3; lipid transfer
 9
C
Pastorello (p.c.)



protein





Pyrus communis
Pyr c 1; Bet v 1
18
C
AF05730


(pear)
homologue






Pyr c 4; profilin
14
C
AF129424



Pyr c 5; isoflavone
  33.5
C
AF071477



transfer protein





Persea americana
Pers a 1; endochitinase
32
C
Z78202


(avocado)






Prunus armeniaca
Pru ar 1; Bet v 1

C
U93165


(apricot)
homologue






Pru ar 3; lipid transfer
 9
P




protein





Prunus avium
Pru av 1; Bet v 1

C
U66076


(sweet cherry)
homologue






Pru av 2; thaumatin

C
U32440



homologue






Pru av 3; lipid transfer
10
C
AF221501



protein






Pru av 4; profilin
15
C
AF129425


Prunus domestica
Pru d 4; lipid transfer
 9
P
119C


(European plum)
protein





Prunus persica
Pru p 3; lipid transfer
10
P
P81402


(peach)
protein






Pru p 4; profilin
14
C
AJ491881


Asparagus
Aspa o 1; lipid transfer
 9
P
119D


officinalis
protein





(asparagus)






Crocus sativus
Cro s 1
21

Varasteh (p.c.)


(saffron crocus)






Lactuca sativa
Lac s 1; lipid transfer
 9

Vieths (p.c.)


(lettuce)
protein





Vitis vinifera
Vit v 1; lipid transfer
 9
P
P80274


(grape)
protein





Musa x
Mus xp 1; profilin
15
C
AF377948


paradisiaca






(banana)






Ananas comosus
Ana c 1; profilin
15
C
AF377949


(pineapple)






Litchi chinensis
Lit c 1; profilin
15
C
AY049013


(litchi)






Sinapis alba
Sin a 1; 2S albumin
14
C
120


(yellow mustard)






Glycine max
Gly m 1; HPS
 7
P
120A


(soybean)
Gly m 2;
 8
P
A57106



Gly m 3; profilin
14
C
AJ223982



Gly m 4; SAM22 PR-10
17
C
120B, X60043



protein





Arachis hypogaea
Ara h 1; vicilin
  63.5
C
L34402


(peanut)
Ara h 2; conglutin
17
C
L77197



Ara h 3; glycinin
60
C
AF093541



Ara h 4; glycinin
37
C
AF086821



Ara h 5; profilin
15
C
AF059616



Ara h 6; conglutin
15
C
AF092846



homologue






Ara h 7; conglutin
15
C
AF091737



homologue






Ara h 8; PR-10 protein
17
C
AY328088


Len culinaris
Len c 1; vicilin
47
C
AJ551424


(lentil)
Len c 2; see biotinylated
66
P
120C



protein





Pisum savitum
Pis s 1; vicilin
44
C
AJ626897


(pea)
Pis s 2; convicilin
63
C
Pending


Actinidia
Act c 1; cysteine protease
30
P
P00785


chinensis
Act c 2; thamautin-like
24
P
121, SW: P81370


(kiwi)
protein





Capsicum
Cap a 1w; osmotin-like
23
C
AJ297410


annuum
protein





(bell pepper)
Cap a 2; profilin
14
C
AJ417552


Lycopersicon
Lyc e 1; profilin
14
C
AJ417553


esculentum
Lyc e 2; fructofuranidase
50
C
AF465612


(tomato)






Solanum
Sola t 1; patatin
43
P
P15476


tuberosum
Sola t 2, cathespin D
21
P
P16348


(potato)
inhibitor






Sola t 3; cysteine protease
21
P
P20347



inhibitor






Sola t 4; aspartic protease
16 + 4
P
P30941



inhibitor





Bertholletia
Ber e 1; 2S albumin
 9
C
P04403, M17146


excelsa
Ber e 2; 11S globulin seed
29
C
AY221641


(Brazil nut)
storage protein





Juglans nigra
Jug n 1; 2S albumin
 19*
C
AY102930


(black walnut)
Jug n 2; vicilin-like
 56*
C
AY102931



protein





Juglans regia
Jug r 1; 2S albumin

C
U66866


(English walnut)
Jug r 2; vicilin
44
C
AF066055



Jug r 3; lipid transfer
 9
P
Pastorello (p.c.)



protein





Anacardium
Ana o 1; vicilin-like
50
C
AF395894


occidentale
protein





(cashew)
Ana o 2; legumin-like
55
C
AF453947



protein






Ana o 3; 2S albumin
14
C
AY081853


Ricinus
Ric c 1; 2S albumin

C
P01089


communis






(Castor bean)






Sesamum
Ses i 1; 2S albumin
 9
C
121A, AF240005


indicum
Ses i 2; 2S albumin
 7
C
AF0911841


(sesame)
Ses i 3; 7S vicilin-like
45
C
AF240006



globulin






Ses i 4; oleosin
17
C
AAG23840



Ses i 5; oleosin
15
C
AAD42924


Cucumis melo
Cuc m 1; serine protease
66
C
D32209


(muskmelon)
Cuc m 2; profilin
14
C
AY271295



Cuc m 3; pathogenesis
 16*
P
P83834



related protein, PR-1
















APPENDIX 9







OTHER ALLERGENS











ALLERGEN
SYSTEMATIC AND

SEQUENCE
ACCESSION NO. OR


SOURCE
ORIGINAL NAMES
MW (KD)
DATA
REFERENCES





Anisakis simplex
Ani s 1
24
P
121B, A59069


(nematode)
Ani s 2; paramyosin
97
C
AF173004



Ani s 3; tropomyosin
41
C
121C, Y19221



Ani s 4
 9
P
P83885


Ascaris suum
Asc s 1
10
P
122


(worm)






Dendronephthya
Den n 1
53
P
122A


nipponica






(soft corral)






Hevea
Hev b 1; elongation
58
P
123, 124


brasiliensis
factor





(rubber)
Hev b 2; 1,3-glucanase
34/36
C
125



Hev b 3
24
P
126, 127



Hev b 4; component of
100-115
P
128



microhelix protein






complex






Hev b 5
16
C
U42640



Hev b 6.01; hevein
20
C
M36986, p02877



precursor






Hev b 6.02; hevein
 5
C
M36986, p02877



Hev b 6.03; C-terminal
14
C
M36986, p02877



fragment






Hev b 7.01; patatin from
42
C
U80598



B-serum homologue






Hev b 7.02, patatin from
44
C
AJ223038



C-serum homologue






Hev b 8; profilin
14
C
Y15042



Hev b 9; enolase
51
C
AJ132580



Hev b 10; Mn-
26
C
AJ249148



superoxide dismutase






Hev b 11; class 1

C
AJ238579



chitinase






Hev b 12; lipid transfer
  9.3
C
AY057860



protein






Hev b 13; esterase
42
P
P83269


Homo sapiens
Hom s 1
 73*
C
Y14314


(human
Hom s 2
  10.3*
C
X80909


autoallergens)
Hom s 3
  20.1*
C
X89985



Hom s 4
 36*
C
Y17711



Hom s 5
  42.6*
C
P02538


Triplochiton
Trip s 1; class 1
  38.5
P
Kespohl (p.c.)


scleroxylon
chitinase





(obeche)









APPENDIX 10—REFERENCES


    1. Marsh, D. G., and L. R. Freidhoff. 1992. ALBE, an allergen database. IUIS, Baltimore, Md., Edition 1.0.
    2. Marsh, D. G., L. Goodfriend, T. P. King, H. Lowenstein, and T. A. E. Platts-Mills. 1986. Allergen nomenclature. Bull WHO 64:767-770.
    3. King, T. P., P. S. Norman, and J. T. Cornell. 1964. Isolation and characterization of allergen from ragweed pollen. II. Biochemistry 3:458-468.
    4. Lowenstein, H. 1980. Timothy pollen allergens. Allergy 35:188-191.
    5. Aukrust, L. 1980. Purification of allergens in Cladosporium herbarum. Allergy 35:206-207.
    6. Demerec, M., E. A. Adelberg, A. J. Clark, and P. E. Hartman. 1966. A proposal for a uniform nomenclature in bacterial genetics. Genetics 54:61-75.
    7. Bodmer, J. G., E. D. Albert, W. F. Bodmer, B. Dupont, H. A. Erlich, B. Mach, S. G. E. Marsh, W. R. Mayr, P. Parham, T. Sasuki, G. M. Th. Schreuder, J. L. Strominger, A. Svejgaard, and P. I. Terasaki. 1991. Nomenclature for factors of the HLA system, 1990. Immunogenetics 33:301-309.
    8. Griffith, I. J., J. Pollock, D. G. Klapper, B. L. Rogers, and A. K. Nault. 1991. Sequence polymorphism of Amb a I and Amb a II, the major allergens in Ambrosia artemisiifolia (short ragweed). Int. Arch. Allergy Appl. Immunol. 96:296-304.
    9. Roebber, M., D. G. Klapper, L. Goodfriend, W. B. Bias, S. H. Hsu, and D. G. Marsh. 1985 Immunochemical and genetic studies of Amb t V (Ra5G), an Ra5 homologue from giant ragweed pollen. J. Immunol. 134:3062-3069.
    10. Metzler, W. J., K. Valentine, M. Roebber, M. Friedrichs, D. G. Marsh, and L. Mueller. 1992. Solution structures of ragweed allergen Amb t V. Biochemistry 31:5117-5127.
    11. Metzler, W. J., K. Valentine, M. Roebber, D. G. Marsh, and L. Mueller. 1992. Proton resonance assignments and three-dimensional solution structure of the ragweed allergen Amb a V by nuclear magnetic resonance spectroscopy. Biochemistry 31:8697-8705.
    12. Goodfriend, L., A. M. Choudhury, J. Del Carpio, and T. P. King. 1979. Cytochromes C: New ragweed pollen allergens. Fed. Proc. 38:1415.
    13. Ekramoddoullah, A. K. M., F. T. Kisil, and A. H. Sehon. 1982. Allergenic cross reactivity of cytochrome c from Kentucky bluegrass and perennial ryegrass pollens. Mol. Immunol. 19:1527-1534.
    14. Ansari, A. A., E. A. Killoran, and D. G. Marsh. 1987. An investigation of human response to perennial ryegrass (Lolium perenne) pollen cytochrome c (Lol p X). J. Allergy Clin. Immunol. 80:229-235.
    15. Morgenstern, J. P., I. J. Griffith, A. W. Brauer, B. L. Rogers, J. F. Bond, M. D. Chapman, and M. Kuo. 1991. Amino acid sequence of Fel d I, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning. Proc. Natl. Acad. Sci. USA 88:9690-9694.
    16. Griffith, I. J., S. Craig, J. Pollock, X. Yu, J. P. Morgenstern, and B. L. Rogers. 1992. Expression and genomic structure of the genes encoding FdI, the major allergen from the domestic cat. Gene 113:263-268.
    17. Weber, A., L. Marz, and F. Altmann. 1986. Characteristics of the asparagine-linked oligosaccharide from honey-bee venom phospholipase A2. Comp. Biochem. Physiol. 83B:321-324.
    18. Weber, A., H. Schroder, K. Thalberg, and L. Marz. 1987. Specific interaction of IgE antibodies with a carbohydrate epitope of honey bee venom phospholipase A2. Allergy 42:464-470.
    19. Stanworth, D. R., K. J. Dorrington, T. E. Hugli, K. Reid, and M. W. Turner. 1990. Nomenclature for synthetic peptides representative of immunoglobulin chain sequences. Bulletin WHO 68:109-111.
    20. Rafnar, T., I. J. Griffith, M. C. Kuo, J. F. Bond, B. L. Rogers, and D. G. Klapper. 1991. Cloning of Amb a I (Antigen E), the major allergen family of short ragweed pollen. J. Biol. Chem. 266: 1229-1236.
    21. Rogers, B. L., J. P. Morgenstern, I. J. Griffith, X. B. Yu, C. M. Counsell, A. W. Brauer, T. P. King, R. D. Garman, and M. C. Kuo. 1991. Complete sequence of the allergen Amb a II: recombinant expression and reactivity with T-cells from ragweed allergic patients. J. Immunol. 147:2547-2552.
    22. Klapper, D. G., L. Goodfriend, and J. D. Capra. 1980 Amino acid sequence of ragweed allergen Ra3. Biochemistry 19:5729-5734.
    23. Ghosh, B., M. P. Perry, T. Rafnar, and D. G. Marsh. 1993. Cloning and expression of immunologically active recombinant Amb a V allergen of short ragweed (Ambrosia artemisiifolia) pollen. J. Immunol. 150:5391-5399.
    24. Roebber, M., R. Hussain, D. G. Klapper, and D. G. Marsh. 1983. Isolation and properties of a new short ragweed pollen allergen, Ra6. J. Immunol. 131:706-711.
    25. Lubahn, B., and D. G. Klapper. 1993. Cloning and characterization of ragweed allergen Amb a VI (abst). J. Allergy Clin. Immunol. 91:338.
    26. Roebber, M., and D. G. Marsh. 1991. Isolation and characterization of allergen Amb a VII from short ragweed pollen. J. Allergy Clin. Immunol. 87:324.
    27. Goodfriend L, Choudhury A M, Klapper D G, Coulter K M, Dorval G, DelCarpio J, Osterland C K. Ra5G, a homologue of Ra5 in giant ragweed pollen: isolation, HLA-DR-associated activity and amino acid sequence. Mol Immunol 22: 899-906, 1985.
    28. Himly M, Jahn-Schmid B, Dedic A, Kelemen P, Wopfner N, Altmann F, van Ree R, Briza P, Richter K, Ebner C, Ferreira F. Art v 1, the major allergen of mugwort pollen, is a modular glycoprotein with a defensin-like and a hydroxyproline-rich domain. FASEB J 17: 106-108, 2003.
    28A. Nilsen, B. M., K. Sletten, M. O'Neill, B. Smestead Paulsen, and H. van Halbeek. 1991. Structural analysis of the glycoprotein allergen Art v II from pollen of mugwort (Artemesia vulgaris). J. Biol. Chem. 266:2660-2668.
    29. Wopfner N, Willeroidee M, Hebenstreit D, van Ree R, Aalbers M, Briza P, Thalhamer J, Ebner C, Richter K, Ferreira F. Molecular and immunological characterization of profilin from mugwort pollen. Biol Chem 383: 1779-1789, 2002.
    29A. Jimenez A, Moreno C, Martinez J, Martinez A, Bartolome B, Guerra F, Palacios R 1994. Sensitization to sunflower pollen: only an occupational allergy? Int Arch Allergy Immunol 105:297-307.
    29B. Barderas R, Villalba M, Lombardero M, Rodriguez R. Identification and characterization of Che a 1 allergen from Chenopodium album pollen. Int Arch Allergy Immunol 127: 47-54, 2002.
    29C. Carnés J, Fernández-Caldas E, Casanovas M, Lahoz C, Colás C. Immunochemical characterization of Salsola kali pollen extracts. Allergy 56, Supplement 68: 274, 2001.
    29D. Giuliani A, Pini C, Bonini S, Mucci N, Ferroni L, Vicari G: Isolation and purification of a major allergen from Parietaria officinalis pollen. Allergy 42: 434-440, 1987.
    30. Smith, P. M., Suphioglu, C., Griffith, I. J., Theriault, K., Knox, R. B. and Singh, M. B. 1996. Cloning and expression in yeast Pichia pastoris of a biologically active form of Cyn d 1, the major allergen of Bermuda grass pollen. J. Allergy Clin. Immunol. 98:331-343.
    31. Suphioglu, C., Ferreira, F. and Knox, R. B. 1997. Molecular cloning and immunological characterisation of Cyn d 7, a novel calcium-binding allergen from Bermuda grass pollen. FEBS Lett. 402:167-172.
    31A. Asturias J A, Arilla M C, Gomez-Bayon N, Martinez J, Martinez A, and Palacios R. 1997. Cloning and high level expression of Cynodon dactylon (Bermuda grass) pollen profilin (Cyn d 12) in Escherichia coli: purification and characterization of the allergen. Clin Exp Allergy 27:1307-1313.
    32. Mecheri, S., G. Peltre, and B. David. 1985. Purification and characterization of a major allergen from Dactylis glomerata pollen: The Ag Dg 1. Int. Arch. Allergy Appl. Immunol. 78:283-289.
    33. Roberts, A. M., L. J. Bevan, P. S. Flora, I. Jepson, and M. R. Walker. 1993. Nucleotide sequence of cDNA encoding the Group II allergen of Cocksfoot/Orchard grass (Dactylis glomerata), Dac g II. Allergy 48:615-623.
    33A. Guerin-Marchand, C., Senechal, H., Bouin, A. P., Leduc-Brodard, V., Taudou, G., Weyer, A., Peltre, G. and David, B. 1996. Cloning, sequencing and immunological characterization of Dac g 3, a major allergen from Dactylis glomerata pollen. Mol. Immunol. 33:797-806.
    34. Klysner, S., K. Welinder, H. Lowenstein, and F. Matthiesen. 1992. Group V allergens in grass pollen IV. Similarities in amino acid compositions and amino terminal sequences of the group V allergens from Lolium perenne, Poa pratensis and Dactylis glomerata. Clin. Exp. Allergy 22: 491-497.
    35. Perez, M., G. Y. Ishioka, L. E. Walker, and R. W. Chesnut. 1990. cDNA cloning and immunological characterization of the rye grass allergen Lol p I. J. Biol. Chem. 265:16210-16215.
    36. Griffith, I. J., P. M. Smith, J. Pollock, P. Theerakulpisut, A. Avjioglu, S. Davies, T. Hough, M. B. Singh, R. J. Simpson, L. D. Ward, and R. B. Knox. 1991. Cloning and sequencing of Lol p I, the major allergenic protein of rye-grass pollen. FEBS Letters 279:210-215.
    37. Ansari, A. A., P. Shenbagamurthi, and D. G. Marsh. 1989. Complete amino acid sequence of a Lolium perenne (perennial rye grass) pollen allergen, Lol p II. J. Biol. Chem. 264:11181-11185.
    37A. Sidoli, A., Tamborini, E., Giuntini, I., Levi, S., Volonte, G., Paini, C., De Lalla, C., Siccardi, A. G., Baralle, F. E., Galliani, S. and Arosio, P. 1993. Cloning, expression, and immunological characterization of recombinant Lolium perenne allergen Lol p II. J. Biol. Chem. 268:21819-21825.
    38. Ansari, A. A., P. Shenbagamurthi, and D. G. Marsh. 1989. Complete primary structure of a Lolium perenne (perennial rye grass) pollen allergen, Lol p III: Comparison with known Lol p I and II sequences. Biochemistry 28:8665-8670.
    39. Singh, M. B., T. Hough, P. Theerakulpisut, A. Avjioglu, S. Davies, P. M. Smith, P. Taylor, R. J. Simpson, L. D. Ward, J. McCluskey, R. Puy, and R. B. Knox. 1991. Isolation of cDNA encoding a newly identified major allergenic protein of rye-grass pollen: Intracellular targeting to the amyloplost. Proc. Natl. Acad. Sci. 88:1384-1388.
    39A. van Ree R, Hoffman D R, van Dijk W, Brodard V, Mahieu K, Koeleman C A, Grande M, van Leeuwen W A, Aalberse R C. 1995. Lol p XI, a new major grass pollen allergen, is a member of a family of soybean trypsin inhibitor-related proteins. J Allergy Clin Immunol 95:970-978.
    40. Suphioglu, C. and Singh, M. B. 1995. Cloning, sequencing and expression in Escherichia coli of Pha a 1 and four isoforms of Pha a 5, the major allergens of canary grass pollen. Clin. Exp. Allergy 25:853-865.
    41. Dolecek, C., Vrtala, S., Laffer, S., Steinberger, P., Kraft, D., Scheiner, O. and Valenta, R. 1993. Molecular characterization of Phl p II, a major timothy grass (Phleum pratense) pollen allergen. FEBS Lett. 335:299-304.
    41A. Fischer S, Grote M, Fahlbusch B, Muller W D, Kraft D, Valenta R. 1996. Characterization of Phl p 4, a major timothy grass (Phleum pratense) pollen allergen. J Allergy Clin Immunol 98:189-198.
    42. Matthiesen, F., and H. Lowenstein. 1991. Group V allergens in grass pollens. I. Purification and characterization of the group V allergen from Phleum pratense pollen, Phl p V. Clin. Exp. Allergy 21:297-307.
    43. Petersen, A., Bufe, A., Schramm, G., Schlaak, M. and Becker, W. M. 1995. Characterization of the allergen group VI in timothy grass pollen (Phl p 6). II. cDNA cloning of Phl p 6 and structural comparison to grass group V. Int. Arch. Allergy Immunol. 108:55-59.
    43A. Marknell DeWitt A, Niederberger V, Lehtonen P, Spitzauer S, Sperr W R, Valent P, Valenta R, Lidholm J. Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11. Clin Exp Allergy 32: 1329-1340, 2002.
    44. Valenta, R., Ball, T., Vrtala, S., Duchene, M., Kraft, D. and Scheiner, O. 1994. cDNA cloning and expression of timothy grass (Phleum pratense) pollen profilin in Escherichia coli: comparison with birch pollen profilin. Biochem. Biophys. Res. Commun. 199:106-118.
    46. Esch, R. E., and D. G. Klapper. 1989. Isolation and characterization of a major cross-reactive grass group I allergenic determinant. Mol. Immunol. 26:557-561.
    47. Olsen, E., L. Zhang, R. D. Hill, F. T. Kisil, A. H. Sehon, and S. Mohapatra. 1991. Identification and characterization of the Poa p IX group of basic allergens of Kentucky bluegrass pollen. J. Immunol. 147:205-211.
    48. Avjioglu, A., M. Singh, and R. B. Knox. 1993. Sequence analysis of Sor h I, the group I allergen of Johnson grass pollen and it comparison to rye-grass Lol p I (abst). J. Allergy Clin. Immunol. 91:340.
    52. Kos T, Hoffmann-Sommergruber K, Ferreira F, Hirschwehr R, Ahorn H, Horak F, Jager S, Sperr W, Kraft D, Scheiner O. 1993. Purification, characterization and N-terminal amino acid sequence of a new major allergen from European chestnut pollen—Cas s 1. Biochem Biophys Res Commun 196:1086-92.
    53. Diaz-Perales A, Lombardero M, Sánchez-Monge R, García-Sellés F J, Pernas M, Fernández-Rivas M, Barber D, Salcedo G. 2000. Lipid-transfer proteins as potential plant panallergens: cross-reactivity among proteins of Artemisia pollen, Castaneae nut and Rosaceae fruits, with different IgE-binding capacities. Clin Exp Allergy 30:1403-1410.
    54. Ipsen, H., and O. C. Hansen. 1991. The NH2-terminal amino acid sequence of the immunochemically partial identical major allergens of alder (Alnus glutinosa) Aln g I, birch (Betula verrucosa) Bet v I, hornbeam (Carpinus betulus) Car b I and oak (Quercus alba) Que a I pollens. Mol. Immunol. 28: 1279-1288.
    55. Taniai, M., S. Ando, M. Usui, M. Kurimoto, M. Sakaguchi, S. Inouye, and T. Matuhasi. 1988. N-terminal amino acid sequence of a major allergen of Japanese cedar pollen (Cry j I). FEBS Lett. 239:329-332.
    56. Griffith, I. J., A. Lussier, R. Garman, R. Koury, H. Yeung, and J. Pollock. 1993. The cDNA cloning of Cry j I, the major allergen of Cryptomeria japonica (Japanese cedar) (abst). J. Allergy Clin. Immunol. 91:339.
    57. Sakaguchi, M., S. Inouye, M. Taniai, S. Ando, M. Usui, and T. Matuhasi. Identification of the second major allergen of Japanese cedar pollen. Allergy 45: 309-312, 1990.
    57A. Yokoyama M, Miyahara M, Shimizu K, Kino K, Tsunoo H. Purification, identification, and cDNA cloning of Jun a 2, the second major allergen of mountain cedar pollen. Biochem Biophys Res Commun 275: 195-202, 2000.
    57B. Midoro-Horiuti T, Goldblum R M, Kurosky A, Wood T G, Brooks E G. Variable Expression of Pathogenesis-Related Protein Allergen in Mountain Cedar (Juniperus ashei) Pollen. J Immunol 164: 2188-2192, 2000.
    57C. Tinghino R., Barletta B., Palumbo S., Afferni C., Iacovacci P., Mari A., Di Felice G., Pini, C. Molecular characterization of a cross-reactive Juniperus oxycedrus pollen allergen, Jun o 2: a novel calcium-binding allergen J. Allergy Clin. Immunol. 101: 772-777, 1998.
    58. Gross G N, Zimburean J M, Capra J D. Isolation and partial characterization of the allergen in mountain cedar pollen. Scand J Immunol 8: 437-441, 1978.
    58a. Obispo T M, Melero J A, Carpizo J A, Carreira J, Lombardero M. The main allergen of Olea europaea (Ole e I) is also present in other species of the oleaceae family. Clin Exp Allergy 23: 311-316, 1993.
    58B. Midoro-Horiuti T, Goldblum R M, Brooks E G. Identification of mutations in the genes for the pollen allergens of eastern red cedar (Juniperus virginiana). Clin Exp Allergy 31: 771-778, 2001.
    59. Lombardero M., Barbas J. A., Moscoso del Prado J., Carreira J. cDNA sequence analysis of the main olive allergen, Ole e I. Clin. Exp. Allergy 24: 765-770, 1994.
    60. Villalba, M., E. Batanero, C. Lopez-Otin, L. M. Sanchez, R. I. Monsalve, M. A. Gonzalez de la Pena, C. Lahoz, and R. Rodriguez Amino acid sequence of Ole e I, the major allergen from olive tree pollen (Olea europaea). Eur. J. Biochem. 216: 863-869, 1993.
    60A. Asturias J A, Arilla M C, Gomez-Bayon N, Martinez J, Martinez A, Palacios R. Cloning and expression of the panallergen profilin and the major allergen (Ole e 1) from olive tree pollen. J Allergy Clin Immunol 100: 365-372, 1997.
    60B. Batanero E, Villalba M, Ledesma A Puente X S, Rodriguez R. Ole e 3, an olive-tree allergen, belongs to a widespread family of pollen proteins. Eur J Biochem 241: 772-778, 1996.
    60C. Batanero E, Ledesma A, Villalba M, Rodriguez R. Purification, amino acid sequence and immunological characterization of Ole e 6, a cysteine-enriched allergen from olive tree pollen. FEBS Lett. 410: 293-296, 1997.
    60D. Tejera M L, Villalba M, Batanero E, Rodriguez R. Identification, isolation, and characterization of Ole e 7, a new allergen of olive tree pollen. J Allergy Clin Immunol 104: 797-802, 1999.
    60E. Ledesma, A., Villalba, M. and Rodriguez, R. Cloning, expression and characterization of a novel four EF-hand Ca(2+)-binding protein from olive pollen with allergenic activity. FEBS Lett. 466: 192-196, 2000.
    61. Yi F C, Cheong N, Shek P C, Wang D Y, Chua K Y, Lee B W. Identification of shared and unique immunoglobulin E epitopes of the highly conserved tropomyosins in Blomia tropicalis and Dermatophagoides pteronyssinus. Clin Exp Allergy 32: 1203-1210, 2002.
    61A. Ramos J D, Cheong N, Lee B W, Chua K Y. cDNA cloning and expression of Blo t 11, the Blomia tropicalis allergen homologous to paramyosin. Int Arch Allergy Immunol 126: 286-293, 2001.
    62. Chua, K. Y., G. A. Stewart, and W. R. Thomas. Sequence analysis of cDNA encoding for a major house dust mite allergen, Der p I. J. Exp. Med. 167: 175-182, 1988.
    62A. Chua, K. Y., C. R. Doyle, R. J. Simpson, K. J. Turner, G. A. Stewart, and W. R. Thomas. Isolation of cDNA coding for the major mite allergen Der p II by IgE plaque immunoassay. Int. Arch. Allergy Appl. Immunol. 91: 118-123, 1990.
    62B. Smith A M, Benjamin D C, Derewenda U, Smith W A, Thomas W R, Chapman M D. Sequence polymorphisms and antibody binding to the group 2 dust mite allergens. Int Arch Allergy Immunol 124: 61-63, 2001.
    62C. Smith A M, Benjamin D C, Hozic N, Derewenda U, Smith W A, Thomas W R, Gafvelin G, van Hage-Hamsten M, Chapman M D. The molecular basis of antigenic cross-reactivity between the group 2 mite allergens. J Allergy Clin Immunol 107: 977-984, 2001.
    63. Smith W A, Thomas W R. Comparative analysis of the genes encoding group 3 allergens from Dermatophagoides pteronyssinus and Dermatophagoides farinae. Int Arch Allergy Immunol 109: 133-140, 1996.
    64. Lake, F. R., L. D. Ward, R. J. Simpson, P. J. Thompson, and G. A. Stewart. House dust mite-derived amylase: Allergenicity and physicochemical characterisation. J. Allergy Clin. Immunol. 87: 1035-1042, 1991.
    65. Tovey, E. R., M. C. Johnson, A. L. Roche, G. S. Cobon, and B. A. Baldo. Cloning and sequencing of a cDNA expressing a recombinant house dust mite protein that binds human IgE and corresponds to an important low molecular weight allergen. J. Exp. Med. 170: 1457-1462, 1989.
    66. Yasueda, H., T. Shida, T. Ando, S. Sugiyama, and H. Yamakawa. 1991. Allergenic and proteolytic properties of fourth allergens from Dermatophagoides mites. In: “Dust Mite Allergens and Asthma. Report of the 2nd international workshop” A. Todt, Ed., UCB Institute of Allergy, Brussels, Belgium, pp. 63-64.
    67. Shen, H.-D., K.-Y. Chua, K.-.L. Lin, K.-H. Hsieh, and W. R. Thomas. Molecular cloning of a house dust mite allergen with common antibody binding specificities with multiple components in mite extracts. Clin. Exp. Allergy 23: 934-940, 1993.
    67. O'Neil G M, Donovan G R, Baldo B A. Cloning and charaterisation of a major allergen of the house dust mite Dermatophagoides pteronyssinus, homologous with glutathione S-transferase. Biochim Biophys Acta, 1219: 521-528, 1994.
    67B. King C, Simpson R J, Moritz R L, Reed G E, Thompson P J, Stewart G A. The isolation and characterization of a novel collagenolytic serine protease allergen (Der p 9) from the dust mite Dermatophagoides pteronyssinus. J Allergy Clin Immunol 98: 739-747, 1996.
    68. Lind P, Hansen O C, Horn N. The binding of mouse hybridoma and human IgE antibodies to the major fecal allergen, Der p I of D. pteronyssinus. J. Immunol. 140: 4256-4262, 1988.
    69. Dilworth, R. J., K. Y. Chua, and W. R. Thomas. Sequence analysis of cDNA coding for a mojor house dust allergen Der f I. Clin. Exp. Allergy 21: 25-32, 1991.
    70. Nishiyama, C., T. Yunki, T. Takai, Y. Okumura, and H. Okudaira. Determination of three disulfide bonds in a major house dust mite allergen, Der f II. Int. Arch. Allergy Immunol. 101: 159-166, 1993.
    70A. Trudinger, M., K. Y. Chua, and W. R. Thomas. cDNA encoding the major dust mite allergen Der f II. Clin. Exp. Allergy 21: 33-38, 1991.
    71. Shen H D, Chua K Y, Lin W L, Hsieh K H, Thomas W R. Molecular cloning and immunological characterization of the house dust mite allergen Der f 7. Clin Exp Allergy 25: 1000-1006, 1995.
    71A. Tategaki A, Kawamoto S, Aki T, Jyo T, Suzuki O, Shigeta S, Ono K. Newly described house dust mite allergens. ACI International suppl. 1: 74-76, 2000.
    72. Aki T, Kodama T, Fujikawa A, Miura K, Shigeta S, Wada T, Jyo T, Murooka Y, Oka S, Ono K Immunochemical characteristion of recombinant and native tropomyosins as a new allergen from the house dust mite Dermatophagoides farinae. J Allergy Clin Immunol 96: 74-83, 1995.
    72A. Tsai L, Sun Y, Chao P, Ng H, Hung M, Hsieh K, Liaw S, Chua K. Sequence analysis and expression of a cDNA clone encoding a 98-kDa allergen in Dermatophagoides farinae. Clin Exp Allergy 29: 1606-1613, 1999.
    72B. Gafvelin G, Johansson E, Lundin A, Smith A M, Chapman M D, Benjamin D C, Derewenda U, Van Hage-Hamsten M. Cross-reactivity studies of a new group 2 allergen from the dust mite Glycyphagus domesticus, Gly d 2, and group 2 allergens from Dermatophagoides pteronyssinus, Lepidoglyphus destructor, and Tyrophagus putrescentiae with recombinant allergens. J Allergy Clin Immunol 107: 511-518, 2001.
    73. van Hage-Hamsten, M., T. Bergman, E. Johansson, B. Persson, H. Jornvall, B. Harfast, and S. G. O. Johansson. N-terminal amino acid sequence of major allergen of the mite lepidoglyphus destructor (abst). J. Allergy Clin. Immunol. 91:353, 1993.
    74. Varela J, Ventas P, Carreira J, Barbas J A, Gimenez-Gallego G, Polo F. Primary structure of Lep d I, the main Lepidoglyphus destructor allergen. Eur J Biochem 225: 93-98, 1994.
    74A. Schmidt M, van der Ploeg I, Olsson S, van Hage Hamsten M. The complete cDNA encoding the Lepidoglyphus destructor major allergen Lep d 1. FEBS Lett 370: 11-14, 1995.
    75. Eriksson T L J, Rasool O, Huecas S, Whitley P, Crameri R, Appenzeller U, Gafvelin G, van Hage-Hamsten M. Cloning of three new allergens from the dust mite Lepidoglyphus destructor using phage surface display technology. Eur. J. Biochem. 268: 287-294, 2001.
    75A. Saarne T, Kaiser L, Rasool O, Huecas S, van Hage-Hamsten M, Gafvelin G: Cloning and characterisation of two IgE-binding proteins, homologous to tropomyosin and a-tubulin, from the mite Lepidoglyphus destructor. Int Arch Allergy Immunol 130: 258-265, 2003.
    75B. Eriksson T L, Johansson E, Whitley P, Schmidt M, Elsayed S, van Hage-Hamsten M. Cloning and characterisation of a group II allergen from the dust mite Tyrophagus putrescentiae. Eur. J. Biochem. 251 (1-2), 443-447, 1998.
    76. Rautiainen J, Rytkonen M, Pelkonen J, Pentikainen J, Perola O, Virtanen T, Zeiler T, Mantyjarvi R. BDA20, a major bovine dander allergen characterized at the sequence level is Bos d 2. Submitted.
    77. Gjesing B, Lowenstein H. Immunochemistry of food antigens. Ann Allergy 53:602, 1984.
    78. de Groot, H., K. G. H. Goei, P. van Swieten, and R. C. Aalberse. Affinity purification of a major and a minor allergen from dog extract: Serologic activity of affinity-purified Can f I and Can f I-depleted extract. J. Allergy Clin. Immunol. 87:1056-1065, 1991.
    79. Konieczny, A. Personal communication; Immunologic Pharmaceutical Corp.
    79A. Bulone, V. Separation of horse dander allergen proteins by two-dimensional electrophoresis. Molecular characterisation and identification of Equ c 2.0101 and Equ c 2.0102 as lipocalin proteins. Eur J Biochem 253: 202-211, 1998.
    79B. Swiss-Prot acc. P81216, P81217.
    79C. Dandeu J. P., Rabillon J., Divanovic A., Carmi-Leroy A., David B. (1993). Hydrophobic interaction chromatography for isolation and purification of Equ c 1, the horse major allergen. J. Chromatogr. 621:23-31.
    79D. Goubran Botros H., Rabillon J., Grégoire C., David B., Dandeu J. P. 1998. Thiophilic absorption chromatography: purification of Equ c 2 and Equ c 3, two horse allergens from horse sweat. J. Chromatogr. B 710:57-65.
    79E. Hilger C, Kohnen M, Grigioni F, Lehners C, Hentges F. Allergic cross-reactions between cat and pig serum albumin. Allergy 52:179-187, 1997; and Hilger C, Grigioni F, Hentges F. Sequence of the gene encoding cat (Felis domesticus) serum albumin. Gene 169:295-296, 1996.
    79F. Ichikawa K, Vailes L D, Pomes A, Chapman M D. Molecular cloning, expression and modeling of cat allergen, cystatin (Fel d 3), a cysteine protease inhibitor. Clin Exp Allergy, In Press 2001.
    80. Fahlbusch B, Rudeschko O, Szilagyi U, Schlott B, Henzgen M, Schlenvoigt G, Schubert H. Purification and partial characterization of the major allergen, Cav p 1, from guinea pig Cavia porcellus. Allergy 57: 417-422, 2002.
    81. McDonald, B., M. C. Kuo, J. L. Ohman, and L. J. Rosenwasser. 1988. A 29 amino acid peptide derived from rat alpha 2 euglobulin triggers murine allergen specific human T-cells (abst). J. Allergy Clin. Immunol. 83:251.
    81A. Clarke, A. J., P. M. Cissold, R. A. Shawi, P. Beattie, and J. Bishop. 1984. Structure of mouse urinary protein genes: differential splicing configurations in the 3′-non-coding region. EMBO J 3:1045-1052.
    82. Longbottom, J. L. 1983. Chracterization of allergens from the urines of experimental animals. McMillan Press, London, pp. 525-529.
    83. Laperche, Y., K. R. Lynch, K. P. Dolans, and P. Feigelsen. 1983. Tissue-specific control of alpha 2u globulin gene expression: constitutive synthesis in submaxillary gland. Cell 32:453-460.
    83A. Bush R K, Sanchez H, Geisler D. 1999. Molecular cloning of a major Alternaria alternata allergen, rAlt a 2. J Allergy Clin Immunol 104:665-671.
    83B. Aukrust L, Borch S M. 1979. Partial purification and characterization of two Cladosporium herbarum allergens. Int Arch Allergy Appl Immunol 60:68-79.
    83C. Sward-Nordmo M, Paulsen B S, Wold J K. 1988. The glycoprotein allergen Ag-54 (Cla h II) from Cladosporium herbarum. Structural studies of the carbohydrate moiety. Int Arch Allergy Appl Immunol 85:288-294.
    84. Shen, et al. J. Allergy Clin. Immunol. 103:S157, 1999.
    84A. Crameri R. Epidemiology and molecular basis of the involvement of Aspergillus fumigatus in allergic diseases. Contrib. Microbiol. Vol. 2, Karger, Basel (in press).
    84B. Shen, et al. (manuscript submitted), 1999
    84C. Shen H D, Ling W L, Tan M F, Wang S R, Chou H, Han S I H. Vacuolar serine proteinase: A major allergen of Aspergillus fumigatus. 10th International Congress of Immunology, Abstract, 1998.
    85. Kumar A, Reddy L V, Sochanik A, Kurup V P. 1993. Isolation and characterization of a recombinant heat shock protein of Aspergillus fumigatus. J. Allergy Clin. Immunol. 91:1024-1030.
    85A. Saxena S, Madan T, Muralidhar K, Sarma P U. 2003. cDNA cloning, expression and characterization of an allergenic L3 ribosomal protein of Aspergillus fumigatus. Clin Exp Immunol 134:86-91.
    86A. Shen H D, Lin W L, Tsai J J, Liaw S F, Han S H. 1996. Allergenic components in three different species of Penicillium: crossreactivity among major allergens. Clin Exp Allergy 26:444-451.
    86B. Shen, et al. Abstract; The XVIII Congress of the European Academy of Allergology and Clinical Immunology, Brussels, Belgium, 3-7 Jul. 1999.
    87. Shen H D, Lin W L, Tam M F, Wang S R, Tzean S S, Huang M H, Han S H. Characterization of allergens from Penicillium oxalicum and P. notatum by immunoblotting and N-terminal amino acid sequence analysis. Clin Exp Allergy 29: 642-651, 1999.
    87A. Shen H D, Liaw S F, Lin W L, Ro L H, Yang H L, Han S H. Molecular cloning of cDNA coding for the 68 kDa allergen of Penicillium notatum using MoAbs. Clin Exp Allergy 25: 350-356, 1995.
    87B. Shen H D, Wang C W, Lin W L, Lai H Y, Tam M F, Chou H, Wang S R, Han S H. cDNA cloning and immunologic characterization of Pen o 18, the vacuolar serine protease major allergen of Penicillium oxalicum. J Lab Clin Med 137: 115-124, 2001.
    88. Woodfolk J A, Wheatley L M, Piyasena R V, Benjamin D C, Platts-Mills T A. 1998. Trichophyton antigens associated with IgE antibodies and delayed type hypersensitivity. Sequence homology to two families of serine proteinases. J Biol Chem 273:29489-96.
    88A. Deuell, B., L. K. Arruda, M. L. Hayden, M. D. Chapman and T. A. E. Platts-Mills. 1991. Trichophyton tonsurans Allergen I. J. Immunol. 147:96-101.
    89. Shen, H. D., K. B. Choo, H. H. Lee, J. C. Hsieh, and S. H. Han. 1991. The 40 kd allergen of Candida albicans is an alcohol dehydrogenease: molecular cloning and immunological analysis using monoclonal antibodies. Clin. Exp. Allergy 21:675-681.
    89A. Homer W E, Reese G, Lehrer S B. 1995. Identification of the allergen Psi c 2 from the basidiomycete Psilocybe cubensis as a fungal cyclophilin. Int Arch Allergy Immunol 107:298-300.
    89B. Chang C Y, Chou H, Tam M F, Tang R B, Lai H Y, Shen H D. Characterization of Enolase Allergen from Rhodotorula mucilaginosa. J Biomed Sci 9: 645-655, 2002.
    90. Yasueda H, Hashida-Okado T, Saito A, Uchida K, Kuroda M, Onishi Y, Takahashi K, Yamaguchi H, Takesako K, Akiyama K. Identification and cloning of two novel allergens from the lipophilic yeast, Malassezia furfur. Biochem Biophys Res Commun 248: 240-244, 1998. NB:strain TIMM2782 (Teikyo University Institute for Medical Mycology) equal to strain CBS1878 (Central Bureau von Schimmelkulturen).
    90A. Onishi Y, Kuroda M, Yasueda H, Saito A, Sono-Koyama E, Tunasawa S, Hashida-Okado T, Yagihara T, Uchida K, Yamaguchi H, Akiyama K, Kato I, Takesako K. Two-dimensional electrophoresis of Malassezia allergens for atopic dermatitis and isolation of Mal f 4 homologs with mitochondrial malate dehydrogenase. Eur J Biochem 261: 148-154, 1999. NB: strain TIMM2782 (Teikyo University Institute for Medical Mycology) equal to strain CBS1878 (Central Bureau von Schimmelkulturen).
    91. Schmidt M, Zargari A, Holt P, Lindbom L, Hellman U, Whitley P, van der Ploeg I, Harfast B, Scheynius A. The complete cDNA sequence and expression of the first major allergenic protein of Malassezia furfur, Mal f 1. Eur J Biochem 246:181-185, 1997. Strain ATCC no. 42132 (American Type Culture Collection).
    91A. Rasool O, Zargari A, Almqvist J, Eshaghi H, Whitley P, Scheynius A. Cloning, characterization and expression of complete coding sequences of three IgE binding Malassezia furfur allergens, Mal f 7, Mal f 8 and Mal f 9. Eur J Biochem 267: 4355-4361, 2000. Strain ATCC no. 42132 (American Type Culture Collection).
    91B. Strain 4625 (Indian Agricultural Research Institute, PUSA; New Delhi, India).
    92. Kuchler, K., M. Gmachl, M. J. Sippl, and G. Kreil. 1989. Analysis of the cDNA for phospholipase A2 from honey bee venom glands: The deduced amino acid sequence reveals homology to the corresponding vertebrate enzymes. Eur. J. Biochem. 184:249-254.
    93. Gmachl, M., and G. Kreil. 1993. Bee venom hyaluronidase is homologous to a membrane protein of mammalian sperm. Proc. Natl. Acad. Sci. USA 90:3569-3573.
    93A. Hoffman D R. 1977. Allergens in bee venom III. Identification of allergen B as an acid phosphatase. J Allergy Clin. Immunol. 59:364-366.
    94. Habermann, E. 1972. Bee and wasp venoms. Science 177:314-322.
    95. Hoffman D R, Jacobson R S. 1996. Allergens in Hymenoptera venom XXVII: Bumblebee venom allergy and allergens. J. Allergy Clin. Immunol. 97:812-821.
    95A. Hoffman D R, El-Choufani A E, Smith M M, de Groot H. 2001. Occupational allergy to bumblebee venom: Allergens of Bombus terrestris. J Allergy Clin Immunol In press.
    95B. Helm R, Cockrell G, Stanley J S, Brenner R J, Burks W, Bannon G A. 1996. Isolation and characterization of a clone encoding a majore allergen (Bla g Bd90K) involved in IgE mediated cockroach hypersensitivity. J Allerg Clin Immunol 98:172-180.
    95C. Pomes A, Melen E, Vailes L D, Retief J D, Arruda L K, Chapman M D. 1998. Novel allergen structures with tandem amino acid repeats derived from German and American cockroach. J Biol Chem 273:30801-30807.
    96. Arruda L K, Vailes L D, Mann B J, Shannon J, Fox J W, Vedvick T S, Hayden M L, Chapman M D. Molecular cloning of a major cockroach (Blattella germanica) allergen, Bla g 2. Sequence homology to the aspartic proteases. J Biol Chem 270:19563-19568, 1995.
    97. Arruda L K, Vailes L D, Hayden M L, Benjamin D C, Chapman M D. Cloning of cockroach allergen, Bla g 4, identifies ligand binding proteins (or calycins) as a cause of IgE antibody responses. J Biol Chem 270:31196-31201, 1995.
    98. Arruda L K, Vailes L D, Benjamin D C, Chapman M D. Molecular cloning of German Cockroach (Blattella germanica) allergens. Int Arch Allergy Immunol 107:295-297, 1995.
    98A. Wu C H, Wang N M, Lee M F, Kao C Y Y, Luo S F. 1998. Cloning of the American cockroach Cr-PII allergens: Evidence for the existence of cross-reactive allergens between species. J Allergy Clin Immunol 101:832-840.
    98B. Melen E, Pomés A, Vailes L D, Arruda L K, Chapman M D. 1999. Molecular cloning of Per a 1 and definition of the cross-reactive Group 1 cockroach allergens. J Allergy Clin Immunol 103:859-64.
    98C. Wu C H, Lee M F, Liao S C, Luo S F. Sequencing analysis of cDNA clones encoding the American cockroach Cr-PI allergens. J Biol Chem 271:17937-17943, 1996.
    98D. Wu C H, Lee M F, Wang N M, Luo S F. Sequencing and immunochemical characterization of the American cockroach Per a 3 (Cr-PI) isoallergenic variants. Molecular Immunol 34:1-8, 1997.
    98E. Santos A B R, Chapman M D, Aalberse R C, Vailes L D, Ferriani V P L, Oliver C, Rizzo M C, Naspitz C K, Arruda L K. 1999. Cockroach allergens and asthma in Brazil: Identification of tropomyosin as a major allergen with potential cross-reactivity with mite and shrimp allergens. J Allergy Clin Immunol 104:329-337.
    98F. Asturias J A, Gómez-Bayón N, Arilla M C, Martinez A, Palacios R, Sánchez-Gascón, Martinez J. 1999. Molecular characterization of American cockroach tropomyosin (Periplaneta americana allergen 7), a cross-reactive allergen. J Immunol 162:4342-4348.
    99. Mazur, G., X. Baur, and V. Liebers. 1990. Hypersensitivity to hemoglobins of the Diptera family Chironomidae: Structural and functional studies of their immunogenic/allergenic sites. Monog. Allergy 28:121-137.
    99A. Moneo I, Vega J M, Caballero M L, Vega J, Alday E. Isolation and characterization of Tha p 1, a major allergen from the pine processionary caterpillar Thaumetopoea pityocampa. Allergy 58: 34-37, 2003.
    100. Soldatova, L., L. Kochoumian, and T. P. King. 1993. Sequence similarity of a hornet (D. maculata) venom allergen phospholipase A1 with mammalian lipases. FEBS Letters 320:145-149.
    101. Lu, G., L. Kochoumian and T. P. King. Whiteface hornet venom allergen hyaluronidase: cloning and its sequence similarity with other proteins (abst.). 1994. J. Allergy Clin. Immunol. 93:224.
    102. Fang, K. S. F., M. Vitale, P. Fehlner, and T. P. King. 1988. cDNA cloning and primary structure of a white-faced hornet venom allergen, antigen 5. Proc. Natl. Acad. Sci., USA 85:895-899.
    103. King, T. P., D. C. Moran, D. F. Wang, L. Kochoumian, and B. T. Chait. 1990. Structural studies of a hornet venom allergen antigen 5, Dol m V and its sequence similarity with other proteins. Prot. Seq. Data Anal. 3:263-266.
    104. Lu, G., M. Villalba, M. R. Coscia, D. R. Hoffman, and T. P. King. 1993. Sequence analysis and antigen cross reactivity of a venom allergen antigen 5 from hornets, wasps and yellowjackets. J. Immunol. 150: 2823-2830.
    105. King, T. P. and Lu, G. 1997. Unpublished data.
    105A. King T P, Lu G, Gonzalez M, Qian N and Soldatova L. 1996. Yellow jacket venom allergens, hyaluronidase and phospholipase: sequence similarity and antigenic cross-reactivity with their hornet and wasp homologs and possible implications for clinical allergy. J. Allergy Clin. Immunol. 98:588-600.
    106. Hoffman, D. R. 1993. Allergens in hymenoptera venom XXV: The amino acid sequences of antigen 5 molecules and the structural basis of antigenic cross-reactivity. J. Allergy Clin. Immunol. 92:707-716.
    107. Hoffman D R. 1992. Unpublished data.
    108. Hoffman D R. The complete amino acid sequence of a yellowjacket venom phospholipase (abst). J. Allergy Clin. Immunol. 91:187, 1993.
    109. Jacobson R S, Hoffman D R, Kemeny D M. The cross-reactivity between bee and vespid hyaluronidases has a structural basis (abst). J. Allergy Clin. Immunol. 89:292, 1992.
    110. Hoffman D R. Allergens in Hymenoptera venom XXIV: The amino acid sequences of imported fire ant venom allergens Sol i II, Sol i III, and Sol i IV. J. Allergy Clin. Immunol 91: 71-78, 1993.
    111. Schmidt M, Walker R B, Hoffman D R, McConnell T J. Nucleotide sequence of cDNA encoding the fire ant venom protein Sol i II. FEBS Letters 319: 138-140, 1993.
    111A. Paddock C D, McKerrow J H, Hansell E, Foreman K W, Hsieh I, Marshall N. Identification, cloning, and recombinant expression of procalin, a major triatomine allergen. J Immunol 167: 2694-2699, 2001.
    112. Elsayed S, Bennich H. The primary structure of Allergen M from cod. Scand J Immunol 3: 683-686, 1974.
    113. Elsayed S, Aas K, Sletten K, Johansson S G O. Tryptic cleavage of a homogeneous cod fish allergen and isolation of two active polypeptide fragments. Immunochemistry 9: 647-661, 1972.
    114. Hoffman, D. R. 1983 Immunochemical identification of the allergens in egg white. J. Allergy Clin. Immunol. 71: 481-486.
    115. Langeland, T. 1983. A clinical and immunological study of allergy to hen's egg white. IV. specific IgE antibodies to individual allergens in hen's egg white related to clinical and immunological parameters in egg-allergic patients. Allergy 38:493-500.
    116 Daul C B, Slattery M, Morgan J E, Lehrer S B. 1993. Common crustacea allergens: identification of B cell epitopes with the shrimp specific monoclonal antibodies. In: “Molecular Biology and Immunology of Allergens” (D. Kraft and A. Sehon, eds.). CRC Press, Boca Raton. pp. 291-293.
    116A Shanti K N, Martin B M, Nagpal S, Metcalfe D D, Subba Rao P V. Identification of tropomyosin as the major shrimp allergen and characterization of its IgE-binding epitopes. J. Immunol. 151: 5354-5363, 1993.
    117. Yu C J, Lin Y F, Chiang B L, Chow L P. Proteomics and Immunological Analysis of a Novel Shrimp Allergen, Pen m 2. J Immunol 170: 445-453, 2003.
    117A Miyazawa M, Fukamachi H, Inagaki Y, Reese G, Daul C B, Lehrer S B, Inouye S, Sakaguchi M. Identification of the first major allergen of a squid (Todarodes pacificus). J. Allergy Clin. Immunol. 98: 948-953, 1996.
    117B. Asturias J A, Eraso E, Arilla M C, Gomez-Bayon N, Inacio F, Martinez A. Cloning, isolation, and IgE-binding properties of Helix aspersa (brown garden snail) tropomyosin. Int Arch Allergy Immunol 128: 90-96, 2002.
    117C. Lopata A L, Zinn C, Potter P C. Characteristics of hypersensitivity reactions and identification of a unique 49 kd IgE-binding protein (Hal-m-1) in abalone (Haliotis midae). J. Allergy Clin. Immunol. 100: 642-648, 1997.
    117D. Hoffmann-Sommergruber K, O'Riordain G, Ahorn H, Ebner C, Laimer Da Camara Machado M, Pühringer H, Scheiner O, Breiteneder H. Molecular characterization of Dau c 1, the Bet v 1 homologous protein from carrot and its cross-reactivity with Bet v 1 and Api g 1. Clin. Exp. Allergy 29: 840-847, 1999.
    118. Monsalve R I, Gonzalez de la Pena M A, Menendez-Arias L, Lopez-Otin C, Villalba M, Rodriguez R. Characterization of a new mustard allergen, Bra j I E. Detection of an allergenic epitope. Biochem. J. 293: 625-632 1993.
    118A. Monsalve R I, Gonzalez de la Pena M A, Lopez-Otin C, Fiandor A, Fernandez C, Villalba M, Rodriguez R. 1997. Detection, isolation and complete amino acid sequence of an aeroallergenic protein from rapeseed flour. Clin Exp Allergy 27:833-841.
    119. Mena, M., R. Sanchez-Monge, L. Gomez, G. Salcedo, and P. Carbonero. A major barley allergen associated with baker's asthma disease is a glycosylated monomeric inhibitor of insect alpha-amylase: cDNA cloning and chromosomal location of the gene. Plant Molec. Biol. 20: 451-458, 1992.
    119A. Palosuo K, Varjonen E, Kekki O M, Klemola T, Kalkkinen N, Alenius H, Reunala T. Wheat omega-5 gliadin is a major allergen in children with immediate allergy to ingested wheat. J. Allergy Clin. Immunol. 108: 634-638, 2001.
    119B. Xu H, Theerakulpisut P, Goulding N, Suphioglu C, Singh M. B. Bhalla P. L. Cloning expression and immunological characterization of Ory s 1, the major allergen of rice pollen. Gene 164: 255-259, 1995.
    119C. Pastorello E A, Ortolani C, Farioli L, Pravettoni V, Ispano M, Borga A, Bengtsson A, Incorvaia C, Berti C, Zanussi C. Allergenic cross-reactivity among peach, apricot, plum, and cherry in patients with oral allergy syndrome: an in vivo and in vitro study. J. Allergy Clin. Immunol. 94: 699-707, 1994.
    119D. Diaz-Perales A, Tabar A I, Sanchez-Monge R, Garcia B E, Gomez B, Barber D, Salcedo G. Characterization of asparagus allergens: a relevant role of lipid transfer proteins. J Allergy Clin Immun ol 110: 790-796, 2002.
    120. Menendez-Arias, L., I. Moneo, J. Dominguez, and R. Rodriguez. 1988. Primary structure of the major allergen of yellow mustard (Sinapis alba L.) seed, Sin a I. Eur. J. Biochem. 177:159-166.
    120A. Gonzalez R, Varela J, Carreira J, Polo F. Soybean hydrophobic protein and soybean hull allergy. Lancet 346:48-49, 1995.
    120B. Kleine-Tebbe J, Vogel L, Crowell D N, Haustein U F, Vieths S. Severe oral allergy syndrome and anaphylactic reactions caused by a Bet v 1-related PR-10 protein in soybean, SAM22. J Allergy Clin Immun ol 110: 797-804, 2002.
    120C. Sanchez-Monge R, Pascual C Y, Diaz-Perales A, Fernandez-Crespo J, Martin-Esteban M, Salcedo G. Isolation and characterization of relevant allergens from boiled lentils. J. Allergy Clin. Immunol. 106: 955-961, 2000.
    121. Gavrovic-Jankulovic M, cIrkovic T, Vuckovic O, Atanaskovic-Markovic M, Petersen A, Gojgic G, Burazer L, Jankov R M. Isolation and biochemical characterization of a thaumatin-like kiwi allergen. J Allergy Clin Immun ol 110: 805-810, 2002.
    121A. Pastorello E A, Varin E, Farioli L, Pravettoni V, Ortolani C, Trambaioli C, Fortunato D, Giuffrida M G, Rivolta F, Robino A, Calamari A M, Lacava L, Conti A. The major allergen of sesame seeds (Sesamum indicum) is a 2S albumin. J. Chromatogr. B Biomed. Sci. Appl. 756: 85-93, 2001.
    121B. Moneo I, Caballero M L, Gomez F, Ortega E, Alonso M J. Isolation and characterization of a major allergen from the fish parasite Anisakis simplex. J. Allergy Clin. Immunol. 106: 177-182, 2000.
    121C. Asturias J A, Eraso E, Martinez A. 2000. Is tropomysoin an allergen in Anisakis? Allergy 55:898-890.
    122. Christie, J. F., B. Dunbar, I. Davidson, and M. W. Kennedy. 1990. N-terminal amino acid sequence identity between a major allergen of Ascaris lumbricoides and Ascaris suum and MHC-restricted IgE responses to it. Immunology 69:596-602.
    122A. Onizuka R, Kamiya H, Muramoto K, Goto R, Inoue K, Kumamoto K, Nakajima Y, Iida S, Ishigami F. Purification of major allergens from red soft coral (Dendrophythya nipponica). Int Arch Allergy Immunol, In press 2001.
    123. Czuppon A B, Chen Z, Rennert S, Engelke T, Meyer H E, Heber M, Baur X. The rubber elongation factor of rubber trees (Hevea brasiliensis) is the major allergen in latex. J Allergy Clin Immunol 92:690-697, 1993.
    124. Attanayaka D P S T G, Kekwick R G O, Franklin F C H. 1991. Molecular cloning and nucleotide sequencing of the rubber elongation factor gene from hevea brasiliensis. Plant Mol Biol 16:1079-1081.
    125. Chye M L, Cheung K Y. 1995. J 1,3-glucanase is highly expressed in Laticifers of Hevea brasiliensis. Plant Mol Biol 26:397-402.
    126. Alenius H, Palosuo T, Kelly K, Kurup V, Reunala T, Makinen-Kiljunen S, Turjanmaa K Fink J. 1993. IgE reactivity to 14-kD and 27-kD natural rubber proteins in Latex-allergic children with Spina bifida and other congenital anomalies. Int Arch Allergy Immunol 102:61-66.
    127. Yeang H Y, Cheong K F, Sunderasan E, Hamzah S, Chew N P, Hamid S, Hamilton R G, Cardosa M J. 1996. The 14.6 kD (REF, Hev b 1) and 24 kD (Hev b 3) rubber particle proteins are recognized by IgE from Spina Bifida patients with Latex allergy. J Allerg Clin Immunol in press.
    128. Sunderasan E, Hamzah S, Hamid S, Ward M A, Yeang H Y, Cardosa M J. 1995. Latex B-serum J-1,3-glucanase (Hev b 2) and a component of the microhelix (Hev b 4) are major Latex allergens. J nat Rubb Res 10:82-99.

## Claims

1. A method of treating allergy in a subject susceptible to an allergic response to a protein allergen, the method comprising a step of:
administering to a subject, via the subject's rectum, a composition comprising:
dead E. coli that has expressed a protein allergen selected from the group consisting of:
Ambrosia artemisiifolia (short ragweed) antigen E (Amb a 1);
Ambrosia artemisiifolia (short ragweed) antigen K (Amb a 2);
Ambrosia artemisiifolia (short ragweed) Ra3 antigen (Amb a 3);
Ambrosia artemisiifolia (short ragweed) Ra5 antigen (Amb a 5);
Ambrosia artemisiifolia (short ragweed) Ra6 antigen (Amb a 6);
Ambrosia artemisiifolia (short ragweed) Ra7 antigen (Amb a 7);
Ambrosia trifida (giant ragweed) Ra5G antigen (Amb t 5);
Artemisia vulgaris (mugwort) antigen (Art v 1);
Artemisia vulgaris (mugwort) antigen (Art v 2);
Helianthus annuus (sunflower) antigen (Hel a 1);
Helianthus annuus (sunflower) profilin (Hel a 2);
Mercurialis annua (annual mercury) profilin (Mer a 1);
Cynodon dactylon (Bermuda grass) antigen (Cyn d 1);
Cynodon dactylon (Bermuda grass) antigen (Cyn d 7);
Cynodon dactylon (Bermuda grass) profilin (Cyn d 12);
Dactylis glomerata (orchard grass) AgDg1 antigen (Dac g 1);
Dactylis glomerata (orchard grass) antigen (Dac g 2);
Dactylis glomerata (orchard grass) antigen (Dac g 3);
Dactylis glomerata (orchard grass) antigen (Dac g 5);
Holcus lanatus (velvet grass) antigen (Hol l 1);
Lolium perenne (rye grass) group I antigen (Lol p 1);
Lolium perenne (rye grass) group II antigen (Lol p 2);
Lolium perenne (rye grass) group III antigen (Lol p 3);
Lolium perenne (rye grass) group IX antigen (Lol p 5);
Lolium perenne (rye grass) antigen (Lol p Ib);
Lolium perenne (rye grass) trypsin (Lol p 11);
Phalaris aquatica (canary grass) antigen (Pha a 1);
Phleum pratense (timothy grass) antigen (Phl p 1);
Phleum pratense (timothy grass) antigen (Phl p 2);
Phleum pratense (timothy grass) antigen (Phl p 4);
Phleum pratense (timothy grass) antigen Ag 25 (Phl p 5);
Phleum pratense (timothy grass) antigen (Phl p 6);
Phleum pratense (timothy grass) profilin (Phl p 12);
Phleum pratense (timothy grass) polygalacturonase (Phl p 13);
Poa pratensis (Kentucky blue grass) group I antigen (Poa p 1);
Poa pratensis (Kentucky blue grass) antigen (Poa p 5);
Sorghum halepense (Johnson grass) antigen (Sor h 1);
Alnus glutinosa (alder) antigen (Aln g 1);
Betula verrucosa (birch) antigen (Bet v 1);
Betula verrucosa (birch) profilin (Bet v 2);
Betula verrucosa (birch) antigen (Bet v 3);
Betula verrucosa (birch) antigen (Bet v 4);
Betula verrucosa (birch) isoflavone reductase homologue (Bet v 5);
Betula verrucosa (birch) cyclophilin (Bet v 7);
Carpinus betulus (hornbeam) antigen (Car b 1);
Castanea sativa (chestnut) Bet v 1 homologue (Cas s 1);
Castanea sativa (chestnut) chitinase (Cas s 5);
Corylus avelana (hazel) antigen (Cor a 1);
Quercus alba (white oak) antigen (Que a 1);
Cryptomeria japonica (sugi) antigen (Cry j 1);
Cryptomeria japonica (sugi) antigen (Cry j 2);
Juniperus ashei (mountain cedar) antigen (Jun a 1);
Juniperus ashei (mountain cedar) antigen (Jun a 3);
Juniperus oxycedrus (prickly juniper) calmodulin-like antigen (Jun o 2);
Juniperus sabinoides (mountain cedar) antigen (Jun s 1);
Juniperus virginiana (eastern red cedar) antigen (Jun v 1);
Fraxinus excelsior (ash) antigen (Fra e 1);
Ligustrum vulgare (privet) antigen (Lig v 1);
Olea europea (olive) antigen (Ole e 1);
Olea europea (olive) profilin (Ole e 2);
Olea europea (olive) antigen (Ole e 3);
Olea europea (olive) antigen (Ole e 4);
Olea europea (olive) superoxide dismutase (Ole e 5);
Olea europea (olive) antigen (Ole e 6);
Syringa vulgaris (lilac) antigen (Syr v 1);
Acarus siro (mite) fatty acid-binding protein (Aca s 13);
Blomia tropicalis (mite) antigen (Blo t 5);
Blomia tropicalis (mite) Bt11a antigen (Blo t 12);
Blomia tropicalis (mite) Bt6 fatty acid-binding protein (Blo t);
Dermatophagoides pteronyssinus (mite) antigen P1 (Der p 1);
Dermatophagoides pteronyssinus (mite) antigen (Der p 2);
Dermatophagoides pteronyssinus (mite) trypsin (Der p 3);
Dermatophagoides pteronyssinus (mite) amylase (Der p 4);
Dermatophagoides pteronyssinus (mite) antigen (Der p 5);
Dermatophagoides pteronyssinus (mite) chymotrypsin (Der p 6);
Dermatophagoides pteronyssinus (mite) antigen (Der p 7);
Dermatophagoides pteronyssinus (mite) glutathione transferase (Der p 8);
Dermatophagoides pteronyssinus (mite) collagenolytic serine prot (Der p 9);
Dermatophagoides pteronyssinus (mite) tropomyosin (Der p 10);
Dermatophagoides pteronyssinus (mite) apolipophorin like p (Der p 14);
Dermatophagoides microceras (mite) antigen (Der m 1);
Dermatophagoides farinae (mite) antigen (Der f 1);
Dermatophagoides farinae (mite) antigen (Der f 2);
Dermatophagoides farinae (mite) antigen (Der f 3);
Dermatophagoides farinae (mite) tropomyosin (Der f 10);
Dermatophagoides farinae (mite) paramyosin (Der f 11);
Dermatophagoides farinae (mite) Mag 3, apolipophorin (Der f 14);
Euroglyphus maynei (mite) apolipophorin (Eur m 14);
Lepidoglyphus destructor (storage mite) antigen (Lep d 2.0101);
Lepidoglyphus destructor (storage mite) antigen (Lep d 2.0102);
Bos domesticus (cow) Ag3, lipocalin (Bos d 2);
Bos domesticus (cow) alpha-lactalbumin (Bos d 4);
Bos domesticus (cow) beta-lactalbumin (Bos d 5);
Bos domesticus (cow) serum albumin (Bos d 6);
Bos domesticus (cow) immunoglobulin (Bos d 7);
Bos domesticus (cow) casein (Bos d 8);
Canis familiaris (dog) antigen (Can f 1);
Canis familiaris (dog) antigen (Can f 2);
Canis familiaris (dog) albumin (Can f ?);
Equus caballus (horse) lipocalin (Equ c 1);
Equus caballus (horse) lipocalin (Equ c 2);
Felis domesticus (cat) cat-1 antigen (Fel d 1);
Mus musculus (mouse) MUP antigen (Mus m 1);
Rattus norvegius (rat) antigen (Rat n 1);
Alternaria alternata (fungus) antigen (Alt a 1);
Alternaria alternata (fungus) antigen (Alt a 2);
Alternaria alternata (fungus) heat shock protein (Alt a 3);
Alternaria alternata (fungus) ribosomal protein (Alt a 6);
Alternaria alternata (fungus) YCP4 protein (Alt a 7);
Alternaria alternata (fungus) aldehyde dehydrogenase (Alt a 10);
Alternaria alternata (fungus) enloase (Alt a 11);
Alternaria alternata (fungus) acid ribosomal protein P1 (Alt a 12);
Cladosporium herbarum (fungus) antigen (Cla h 1);
Cladosporium herbarum (fungus) antigen (Cla h 2);
Cladosporium herbarum (fungus) aldehyde dehydrogenase (Cla h 3);
Cladosporium herbarum (fungus) ribosomal protein);
Cladosporium herbarum (fungus) YCP4 protein (Cla h 5);
Cladosporium herbarum (fungus) enolase (Cla h 6);
Cladosporium herbarum (fungus) acid ribosomal protein P1 (Cla h 12);
Aspergillus flavus (fungus) alkaline serine proteinase (Asp fl 13);
Aspergillus Fumigatus (fungus) antigen (Asp f 1);
Aspergillus Fumigatus (fungus) antigen (Asp f 2);
Aspergillus Fumigatus (fungus) peroxisomal protein (Asp f 3);
Aspergillus Fumigatus (fungus) antigen (Asp f 4);
Aspergillus Fumigatus (fungus) metalloprotease (Asp f 5);
Aspergillus Fumigatus (fungus) Mn superoxide dismutase (Asp f 6);
Aspergillus Fumigatus (fungus) antigen (Asp f 7);
Aspergillus Fumigatus (fungus) ribosomal protein P2 (Asp f 8);
Aspergillus Fumigatus (fungus) antigen (Asp f 9);
Aspergillus Fumigatus (fungus) aspartis protease (Asp f 10);
Aspergillus Fumigatus (fungus) peptidyl-prolyl isomerase (Asp f 11);
Aspergillus Fumigatus (fungus) heat shock protein P70 (Asp f 12);
Aspergillus Fumigatus (fungus) alkaline serine proteinase (Asp f 13);
Aspergillus Fumigatus (fungus) antigen (Asp f 15);
Aspergillus Fumigatus (fungus) antigen (Asp f 16);
Aspergillus Fumigatus (fungus) antigen (Asp f 17);
Aspergillus Fumigatus (fungus) vacuolar serine (Asp f 18);
Aspergillus niger (fungus) beta-xylosidase (Asp n 14);
Aspergillus niger (fungus) antigen (Asp n 18);
Aspergillus niger (fungus) vacuolar serine proteinase;
Aspergillus oryzae (fungus) TAKA-amylase A (Asp o 2);
Aspergillus oryzae (fungus) alkaline serine proteinase (Asp o 13);
Penicillium brevicompactum (fungus) alkaline serine proteinase (Pen b 13);
Penicillium citrinum (fungus) heat shock protein P70 (Pen c 1);
Penicillium citrinum (fungus) peroxisomal membrane protein (Pen c 3);
Penicillium citrinum (fungus) alkaline serine proteinase (Pen c 13);
Penicillium notatum (fungus) N-acetyl glucosaminidase (Pen n 1);
Penicillium notatum (fungus) alkaline serine proteinase (Pen n 13);
Penicillium notatum (fungus) vacuolar serine proteinase (Pen n 18);
Penicillium oxalicum (fungus) vacuolar serine proteinase (Pen o 18);
Trichophyton rubrum (fungus) antigen (Tri r 2);
Trichophyton rubrum (fungus) serine protease (Tri r 4);
Trichophyton tonsurans (fungus) antigen (Tri t 1);
Trichophyton tonsurans (fungus) serine protease (Tri t 4);
Candida albicans (fungus) antigen (Cand a 1);
Candida boidinii (fungus) antigen (Cand b 2);
Malassezia furfur (fungus) antigen (Mal f 1);
Malassezia furfur (fungus) MF1 peroxisomal membrane protein (Mal f 2);
Malassezia furfur (fungus) MF2 peroxisomal membrane protein (Mal f 3);
Malassezia furfur (fungus) antigen (Mal f 4);
Malassezia furfur (fungus) antigen (Mal f 5);
Malassezia furfur (fungus) cyclophilin homologue (Mal f 6);
Psilocybe cubensis (fungus) antigen (Psi c 1);
Psilocybe cubensis (fungus) cyclophilin (Psi c 2);
Coprinus comatus (shaggy cap) antigen (Cop c 1);
Coprinus comatus (shaggy cap) antigen (Cop c 2);
Coprinus comatus (shaggy cap) antigen (Cop c 3);
Coprinus comatus (shaggy cap) antigen (Cop c 5);
Coprinus comatus (shaggy cap) antigen (Cop c 7);
Aedes aegyptii (mosquito) apyrase (Aed a 1);
Aedes aegyptii (mosquito) antigen (Aed a 2);
Apis mellifera (honey bee) phospholipase A2 (Api m 1);
Apis mellifera (honey bee) hyaluronidase (Api m 2);
Apis mellifera (honey bee) melittin (Api m 4);
Apis mellifera (honey bee) antigen (Api m 6);
Bombus pennsylvanicus (bumble bee) phospholipase (Bom p 1);
Bombus pennsylvanicus (bumble bee) protease (Bom p 4);
Blattella germanica (German cockroach) Bd90k (Bla g 1);
Blattella germanica (German cockroach) aspartic protease (Bla g 2);
Blattella germanica (German cockroach) calycin (Bla g 4);
Blattella germanica (German cockroach) glutathione transferase (Bla g 5);
Blattella germanica (German cockroach) troponin C (Bla g 6);
Periplaneta americana (American cockroach) Cr-PII (Per a 1);
Periplaneta americana (American cockroach) Cr-PI (Per a 3);
Periplaneta americana (American cockroach) tropomyosin (Per a 7);
Chironomus thummi thummi (midge) hemoglobin (Chi t 1-9);
Chironomus thummi thummi (midge) component III (Chi t 1.01);
Chironomus thummi thummi (midge) component IV (Chi t 1.02);
Chironomus thummi thummi (midge) component I (Chi t 2.0101);
Chironomus thummi thummi (midge) component IA (Chi t 2.0102);
Chironomus thummi thummi (midge) component II-beta (Chi t 3);
Chironomus thummi thummi (midge) component IIIA (Chi t 4);
Chironomus thummi thummi (midge) component VI (Chi t 5);
Chironomus thummi thummi (midge) component VIIA (Chi t 6.01);
Chironomus thummi thummi (midge) component IX (Chi t 6.02);
Chironomus thummi thummi (midge) component VIIB (Chi t 7);
Chironomus thummi thummi (midge) component VIII (Chi t 8);
Chironomus thummi thummi (midge) component X (Chi t 9);
Dolichovespula maculata (white face hornet) phospholipase (Dol m 1);
Dolichovespula maculata (white face hornet) hyaluronidase (Dol m 2);
Dolichovespula maculata (white face hornet) antigen 5 (Dol m 5);
Dolichovespula arenaria (yellow hornet) antigen 5 (Dol a 5);
Polistes annularies (wasp) phospholipase A1 (Pol a 1);
Polistes annularies (wasp) hyaluronidase (Pol a 2);
Polistes annularies (wasp) antigen 5 (Pol a 5);
Polistes dominulus (Mediterranean paper wasp) antigen (Pol d 1);
Polistes dominulus (Mediterranean paper wasp) serine protease (Pol d 4);
Polistes dominulus (Mediterranean paper wasp) antigen (Pol d 5);
Polistes exclamans (wasp) phospholipase A1 (Pol e 1);
Polistes exclamans (wasp) antigen 5 (Pol e 5);
Polistes fuscatus (wasp) antigen 5 (Pol f 5);
Polistes metricus (wasp) antigen 5 (Pol m 5);
Vespa crabo (European hornet) phospholipase (Vesp c 1);
Vespa crabo (European hornet) antigen 5 (Vesp c 5.0101);
Vespa crabo (European hornet) antigen 5 (Vesp c 5.0102);
Vespa mandarina (giant Asian hornet) antigen (Vesp m 1.01);
Vespa mandarina (giant Asian hornet) antigen (Vesp m 1.02);
Vespa mandarina (giant Asian hornet) antigen (Vesp m 5);
Vespula flavopilosa (yellowjacket) antigen 5 (Ves f 5);
Vespula germanica (yellowjacket) antigen 5 (Ves g 5);
Vespula maculifrons (yellowjacket) phospholipase A1 (Ves m 1);
Vespula maculifrons (yellowjacket) hyaluronidase (Ves m 2);
Vespula maculifrons (yellowjacket) antigen 5 (Ves m 5);
Vespula pennsylvanica (yellowjacket) (antigen 5Ves p 5);
Vespula squamosa (yellowjacket) antigen 5 (Ves s 5);
Vespula vidua (wasp) antigen (Ves vi 5);
Vespula vulgaris (yellowjacket) phospholipase A1 (Ves v 1);
Vespula vulgaris (yellowjacket) hyaluronidase (Ves v 2);
Vespula vulgaris (yellowjacket) antigen 5 (Ves v 5);
Myrmecia pilosula (Australian jumper ant) antigen (Myr p 1);
Myrmecia pilosula (Australian jumper ant) antigen (Myr p 2);
Solenopsis geminata (tropical fire ant) antigen (Sol g 2);
Solenopsis geminata (tropical fire ant) antigen (Sol g 4);
Solenopsis invicta (fire ant) antigen (Sol i 2);
Solenopsis invicta (fire ant) antigen (Sol i 3);
Solenopsis invicta (fire ant) antigen (Sol i 4);
Solenopsis saevissima (Brazilian fire ant) antigen (Sol s 2);
Gadus callarias (cod) allergen M (Gad c 1);
Salmo salar (Atlantic salmon) parvalbumin (Sal s 1);
Gallus domesticus (chicken) ovomucoid (Gal d 1);
Gallus domesticus (chicken) ovalbumin (Gal d 2);
Gallus domesticus (chicken) conalbumin; A22 (Gal d 3);
Gallus domesticus (chicken) lysozyme (Gal d 4);
Gallus domesticus (chicken) serum albumin (Gal d 5);
Metapenaeus ensis (shrimp) tropomyosin (Met e 1);
Penaeus aztecus (shrimp) tropomyosin (Pen a 1);
Penaeus indicus (shrimp) tropomyosin (Pen i 1);
Todarodes pacificus (squid) tropomyosin (Tod p 1);
Haliotis Midae (abalone) antigen (Hal m 1);
Apium graveolens (celery) Bet v 1 homologue (Api g 1);
Apium graveolens (celery) profilin (Api g 4);
Apium graveolens (celery) antigen (Api g 5);
Brassica juncea (oriental mustard) 2S albumin (Bra j 1);
Brassica rapa (turnip) prohevein-like protein (Bar r 2);
Hordeum vulgare (barley) BMAI-1 (Hor v 1);
Zea mays (maize, corn) lipid transfer protein (Zea m 14);
Corylus avellana (hazelnut) Bet v 1 homologue (Cor a 1.0401);
Malus domestica (apple) Bet v 1 homologue (Mal d 1);
Malus domestica (apple) lipid transfer protein (Mal d 3);
Pyrus communis (pear) Bet v 1 homologue (Pyr c 1);
Pyrus communis (pear) profilin (Pyr c 4);
Pyrus communis (pear) isoflavone reductase homologue (Pyr c 5);
Oryza sativa (rice) antigen (Ory s 1);
Persea americana (avocado) endochitinase (Pers a 1);
Prunus armeniaca (apricot) Bet v 1 homologue (Pru ar 1);
Prunus armeniaca (apricot) lipid transfer protein (Pru ar 3);
Prunus avium (sweet cherry) Bet v 1 homologue (Pru av 1);
Prunus avium (sweet cherry) thaumatin homologue (Pru av 2);
Prunus avium (sweet cherry) profilin (Pru av 4);
Prunus persica (peach) lipid transfer protein (Pru p 3);
Sinapis alba (yellow mustard) 2S albumin (Sin a 1);
Glycine max (soybean) HPS (Gly m 1.0101);
Glycine max (soybean) HPS (Gly m 1.0102);
Glycine max (soybean) antigen (Gly m 2);
Glycine max (soybean) profilin (Gly m 3);
Arachis hypogaea (peanut) vicilin (Ar a h 1);
Arachis hypogaea (peanut) (conglutin Ar a h 2);
Arachis hypogaea (peanut) glycinin (Ar a h 3);
Arachis hypogaea (peanut) glycinin (Ar a h 4);
Arachis hypogaea (peanut) (profilin Ar a h 5);
Arachis hypogaea (peanut) conglutin homologue (Ar a h 6);
Arachis hypogaea (peanut) conglutin homologue (Ar a h 7);
Actinidia chinensis (kiwi) cysteine protease (Act c 1);
Solanum tuberosum (potato) patatin (Sol t 1);
Bertholletia excelsa (Brazil nut) 2S albumin (Ber e 1);
Juglans regia (English walnut) 2S albumin (Jug r 1);
Juglans regia (English walnut) vicilin (Jug r 2);
Ricinus communis (castor bean) 2S albumin (Ric c 1);
Anisakis simplex (nematode) antigen (Ani s 1);
Anisakis simplex (nematode) paramyosin (Ani s 2);
Ascaris suum (worm) antigen (Asc s 1);
Aedes aegyptii (mosquito) apyrase (Aed a 1);
Aedes aegyptii (mosquito) antigen (Aed a 2);
Hevea brasiliensis (rubber) elongation factor (Hev b 1);
Hevea brasiliensis (rubber) 1,3-glucanase (Hev b 2);
Hevea brasiliensis (rubber) antigen (Hev b 3);
Hevea brasiliensis (rubber) component of microhelix protein complex (Hev b 4);
Hevea brasiliensis (rubber) antigen (Hev b 5);
Hevea brasiliensis (rubber) hevein precursor (Hev b 6.01);
Hevea brasiliensis (rubber) hevein (Hev b 6.02);
Hevea brasiliensis (rubber) C-terminal fragment antigen (Hev b 6.03);
Hevea brasiliensis (rubber) patatin homologue (Hev b 7);
Hevea brasiliensis (rubber) profilin (Hev b 8);
Hevea brasiliensis (rubber) enolase (Hev b 9);
Hevea brasiliensis (rubber) Mn-superoxide dismut (Hev b 10);
Ctenocephalides felis felis (cat flea) antigen (Cte f 1);
Homo sapiens (human autoallergen) antigen (Hom s 1);
Homo sapiens (human autoallergen) antigen (Hom s 2);
Homo sapiens (human autoallergen) antigen (Hom s 3);
Homo sapiens (human autoallergen) antigen (Hom s 4); and
Homo sapiens (human autoallergen) antigen (Hom s 5),
the expressed protein allergen being encapsulated within the dead E. coli, so that the expressed protein allergen is not exposed to the subject's IgE antibodies during administration; and
a pharmaceutically acceptable carrier,


so that one or more symptoms of the subject's allergy to the protein allergen is reduced or decreased.

2. A method of treating allergy in a subject susceptible to an allergic response to a protein allergen, the method comprising a step of:
administering to a subject, via the subject's rectum, a composition comprising:
dead E. coli that has expressed a protein allergen selected from the group consisting of:
Ambrosia artemisiifolia (short ragweed) antigen E (Amb a 1);
Ambrosia artemisiifolia (short ragweed) antigen K (Amb a 2);
Ambrosia artemisiifolia (short ragweed) Ra3 antigen (Amb a 3);
Ambrosia artemisiifolia (short ragweed) Ra5 antigen (Amb a 5);
Ambrosia artemisiifolia (short ragweed) Ra6 antigen (Amb a 6);
Ambrosia artemisiifolia (short ragweed) Ra7 antigen (Amb a 7);
Ambrosia trifida (giant ragweed) Ra5G antigen (Amb t 5);
Artemisia vulgaris (mugwort) antigen (Art v 1);
Artemisia vulgaris (mugwort) antigen (Art v 2);
Helianthus annuus (sunflower) antigen (Hel a 1);
Helianthus annuus (sunflower) profilin (Hel a 2);
Mercurialis annua (annual mercury) profilin (Mer a 1);
Cynodon dactylon (Bermuda grass) antigen (Cyn d 1);
Cynodon dactylon (Bermuda grass) antigen (Cyn d 7);
Cynodon dactylon (Bermuda grass) profilin (Cyn d 12);
Dactylis glomerata (orchard grass) AgDg1 antigen (Dac g 1);
Dactylis glomerata (orchard grass) antigen (Dac g 2);
Dactylis glomerata (orchard grass) antigen (Dac g 3);
Dactylis glomerata (orchard grass) antigen (Dac g 5);
Holcus lanatus (velvet grass) antigen (Hol l 1);
Lolium perenne (rye grass) group I antigen (Lol p 1);
Lolium perenne (rye grass) group II antigen (Lol p 2);
Lolium perenne (rye grass) group III antigen (Lol p 3);
Lolium perenne (rye grass) group IX antigen (Lol p 5);
Lolium perenne (rye grass) antigen (Lol p Ib);
Lolium perenne (rye grass) trypsin (Lol p 11);
Phalaris aquatica (canary grass) antigen (Pha a 1);
Phleum pratense (timothy grass) antigen (Phl p 1);
Phleum pratense (timothy grass) antigen (Phl p 2);
Phleum pratense (timothy grass) antigen (Phl p 4);
Phleum pratense (timothy grass) antigen Ag 25 (Phl p 5);
Phleum pratense (timothy grass) antigen (Phl p 6);
Phleum pratense (timothy grass) profilin (Phl p 12);
Phleum pratense (timothy grass) polygalacturonase (Phl p 13);
Poa pratensis (Kentucky blue grass) group I antigen (Poa p 1);
Poa pratensis (Kentucky blue grass) antigen (Poa p 5);
Sorghum halepense (Johnson grass) antigen (Sor h 1);
Alnus glutinosa (alder) antigen (Aln g 1);
Betula verrucosa (birch) antigen (Bet v 1);
Betula verrucosa (birch) profilin (Bet v 2);
Betula verrucosa (birch) antigen (Bet v 3);
Betula verrucosa (birch) antigen (Bet v 4);
Betula verrucosa (birch) isoflavone reductase homologue (Bet v 5);
Betula verrucosa (birch) cyclophilin (Bet v 7);
Carpinus betulus (hornbeam) antigen (Car b 1);
Castanea sativa (chestnut) Bet v 1 homologue (Cas s 1);
Castanea sativa (chestnut) chitinase (Cas s 5);
Corylus avelana (hazel) antigen (Cor a 1);
Quercus alba (white oak) antigen (Que a 1);
Cryptomeria japonica (sugi) antigen (Cry j 1);
Cryptomeria japonica (sugi) antigen (Cry j 2);
Juniperus ashei (mountain cedar) antigen (Jun a 1);
Juniperus ashei (mountain cedar) antigen (Jun a 3);
Juniperus oxycedrus (prickly juniper) calmodulin-like antigen (Jun o 2);
Juniperus sabinoides (mountain cedar) antigen (Jun s 1);
Juniperus virginiana (eastern red cedar) antigen (Jun v 1);
Fraxinus excelsior (ash) antigen (Fra e 1);
Ligustrum vulgare (privet) antigen (Lig v 1);
Olea europea (olive) antigen (Ole e 1);
Olea europea (olive) profilin (Ole e 2);
Olea europea (olive) antigen (Ole e 3);
Olea europea (olive) antigen (Ole e 4);
Olea europea (olive) superoxide dismutase (Ole e 5);
Olea europea (olive) antigen (Ole e 6);
Syringa vulgaris (lilac) antigen (Syr v 1);
Acarus siro (mite) fatty acid-binding protein (Aca s 13);
Blomia tropicalis (mite) antigen (Blo t 5);
Blomia tropicalis (mite) Bt11a antigen (Blo t 12);
Blomia tropicalis (mite) Bt6 fatty acid-binding protein (Blo t);
Dermatophagoides pteronyssinus (mite) antigen P1 (Der p 1);
Dermatophagoides pteronyssinus (mite) antigen (Der p 2);
Dermatophagoides pteronyssinus (mite) trypsin (Der p 3);
Dermatophagoides pteronyssinus (mite) amylase (Der p 4);
Dermatophagoides pteronyssinus (mite) antigen (Der p 5);
Dermatophagoides pteronyssinus (mite) chymotrypsin (Der p 6);
Dermatophagoides pteronyssinus (mite) antigen (Der p 7);
Dermatophagoides pteronyssinus (mite) glutathione transferase (Der p 8);
Dermatophagoides pteronyssinus (mite) collagenolytic serine prot. (Der p 9);
Dermatophagoides pteronyssinus (mite) tropomyosin (Der p 10);
Dermatophagoides pteronyssinus (mite) apolipophorin like p (Der p 14);
Dermatophagoides microceras (mite) antigen (Der m 1);
Dermatophagoides farinae (mite) antigen (Der f 1);
Dermatophagoides farinae (mite) antigen (Der f 2);
Dermatophagoides farinae (mite) antigen (Der f 3);
Dermatophagoides farinae (mite) tropomyosin (Der f 10);
Dermatophagoides farinae (mite) paramyosin (Der f 11);
Dermatophagoides farinae (mite) Mag 3, apolipophorin (Der f 14);
Euroglyphus maynei (mite) apolipophorin (Eur m 14);
Lepidoglyphus destructor (storage mite) antigen (Lep d 2.0101);
Lepidoglyphus destructor (storage mite) antigen (Lep d 2.0102);
Bos domesticus (cow) Ag3, lipocalin (Bos d 2);
Bos domesticus (cow) alpha-lactalbumin (Bos d 4);
Bos domesticus (cow) beta-lactalbumin (Bos d 5);
Bos domesticus (cow) serum albumin (Bos d 6);
Bos domesticus (cow) immunoglobulin (Bos d 7);
Bos domesticus (cow) casein (Bos d 8);
Canis familiaris (dog) antigen (Can f 1);
Canis familiaris (dog) antigen (Can f 2);
Canis familiaris (dog) albumin (Can f ?);
Equus caballus (horse) lipocalin (Equ c 1);
Equus caballus (horse) lipocalin (Equ c 2);
Felis domesticus (cat) cat-1 antigen (Fel d 1);
Mus musculus (mouse) MUP antigen (Mus m 1);
Rattus norvegius (rat) antigen (Rat n 1);
Alternaria alternata (fungus) antigen (Alt a 1);
Alternaria alternata (fungus) antigen (Alt a 2);
Alternaria alternata (fungus) heat shock protein (Alt a 3);
Alternaria alternata (fungus) ribosomal protein (Alt a 6);
Alternaria alternata (fungus) YCP4 protein (Alt a 7);
Alternaria alternata (fungus) aldehyde dehydrogenase (Alt a 10);
Alternaria alternata (fungus) enloase (Alt a 11);
Alternaria alternata (fungus) acid ribosomal protein P1 (Alt a 12);
Cladosporium herbarum (fungus) antigen (Cla h 1);
Cladosporium herbarum (fungus) antigen (Cla h 2);
Cladosporium herbarum (fungus) aldehyde dehydrogenase (Cla h 3);
Cladosporium herbarum (fungus) ribosomal protein);
Cladosporium herbarum (fungus) YCP4 protein (Cla h 5);
Cladosporium herbarum (fungus) enolase (Cla h 6);
Cladosporium herbarum (fungus) acid ribosomal protein P1 (Cla h 12);
Aspergillus flavus (fungus) alkaline serine proteinase (Asp fl 13);
Aspergillus Fumigatus (fungus) antigen (Asp f 1);
Aspergillus Fumigatus (fungus) antigen (Asp f 2);
Aspergillus Fumigatus (fungus) peroxisomal protein (Asp f 3);
Aspergillus Fumigatus (fungus) antigen (Asp f 4);
Aspergillus Fumigatus (fungus) metalloprotease (Asp f 5);
Aspergillus Fumigatus (fungus) Mn superoxide dismutase (Asp f 6);
Aspergillus Fumigatus (fungus) antigen (Asp f 7);
Aspergillus Fumigatus (fungus) ribosomal protein P2 (Asp f 8);
Aspergillus Fumigatus (fungus) antigen (Asp f 9);
Aspergillus Fumigatus (fungus) aspartis protease (Asp f 10);
Aspergillus Fumigatus (fungus) peptidyl-prolyl isomerase (Asp f 11);
Aspergillus Fumigatus (fungus) heat shock protein P70 (Asp f 12);
Aspergillus Fumigatus (fungus) alkaline serine proteinase (Asp f 13);
Aspergillus Fumigatus (fungus) antigen (Asp f 15);
Aspergillus Fumigatus (fungus) antigen (Asp f 16);
Aspergillus Fumigatus (fungus) antigen (Asp f 17);
Aspergillus Fumigatus (fungus) vacuolar serine (Asp f 18);
Aspergillus niger (fungus) beta-xylosidase (Asp n 14);
Aspergillus niger (fungus) antigen (Asp n 18);
Aspergillus niger (fungus) vacuolar serine proteinase;
Aspergillus oryzae (fungus) TAKA-amylase A (Asp o 2);
Aspergillus oryzae (fungus) alkaline serine proteinase (Asp o 13);
Penicillium brevicompactum (fungus) alkaline serine proteinase (Pen b 13);
Penicillium citrinum (fungus) heat shock protein P70 (Pen c 1);
Penicillium citrinum (fungus) peroxisomal membrane protein (Pen c 3);
Penicillium citrinum (fungus) alkaline serine proteinase (Pen c 13);
Penicillium notatum (fungus) N-acetyl glucosaminidase (Pen n 1);
Penicillium notatum (fungus) alkaline serine proteinase (Pen n 13);
Penicillium notatum (fungus) vacuolar serine proteinase (Pen n 18);
Penicillium oxalicum (fungus) vacuolar serine proteinase (Pen o 18);
Trichophyton rubrum (fungus) antigen (Tri r 2);
Trichophyton rubrum (fungus) serine protease (Tri r 4);
Trichophyton tonsurans (fungus) antigen (Tri t 1);
Trichophyton tonsurans (fungus) serine protease (Tri t 4);
Candida albicans (fungus) antigen (Cand a 1);
Candida boidinii (fungus) antigen (Cand b 2);
Malassezia furfur (fungus) antigen (Mal f 1);
Malassezia furfur (fungus) MF1 peroxisomal membrane protein (Mal f 2);
Malassezia furfur (fungus) MF2 peroxisomal membrane protein (Mal f 3);
Malassezia furfur (fungus) antigen (Mal f 4);
Malassezia furfur (fungus) antigen (Mal f 5);
Malassezia furfur (fungus) cyclophilin homologue (Mal f 6);
Psilocybe cubensis (fungus) antigen (Psi c 1);
Psilocybe cubensis (fungus) cyclophilin (Psi c 2);
Coprinus comatus (shaggy cap) antigen (Cop c 1);
Coprinus comatus (shaggy cap) antigen (Cop c 2);
Coprinus comatus (shaggy cap) antigen (Cop c 3);
Coprinus comatus (shaggy cap) antigen (Cop c 5);
Coprinus comatus (shaggy cap) antigen (Cop c 7);
Aedes aegyptii (mosquito) apyrase (Aed a 1);
Aedes aegyptii (mosquito) antigen (Aed a 2);
Apis mellifera (honey bee) phospholipase A2 (Api m 1);
Apis mellifera (honey bee) hyaluronidase (Api m 2);
Apis mellifera (honey bee) melittin (Api m 4);
Apis mellifera (honey bee) antigen (Api m 6);
Bombus pennsylvanicus (bumble bee) phospholipase (Bom p 1);
Bombus pennsylvanicus (bumble bee) protease (Bom p 4);
Blattella germanica (German cockroach) Bd90k (Bla g 1);
Blattella germanica (German cockroach) aspartic protease (Bla g 2);
Blattella germanica (German cockroach) calycin (Bla g 4);
Blattella germanica (German cockroach) glutathione transferase (Bla g 5);
Blattella germanica (German cockroach) troponin C (Bla g 6);
Periplaneta americana (American cockroach) Cr-PII (Per a 1);
Periplaneta americana (American cockroach) Cr-PI (Per a 3);
Periplaneta americana (American cockroach) tropomyosin (Per a 7);
Chironomus thummi thummi (midge) hemoglobin (Chi t 1-9);
Chironomus thummi thummi (midge) component III (Chi t 1.01);
Chironomus thummi thummi (midge) component IV (Chi t 1.02);
Chironomus thummi thummi (midge) component I (Chi t 2.0101);
Chironomus thummi thummi (midge) component IA (Chi t 2.0102);
Chironomus thummi thummi (midge) component II-beta (Chi t 3);
Chironomus thummi thummi (midge) component IIIA (Chi t 4);
Chironomus thummi thummi (midge) component VI (Chi t 5);
Chironomus thummi thummi (midge) component VIIA (Chi t 6.01);
Chironomus thummi thummi (midge) component IX (Chi t 6.02);
Chironomus thummi thummi (midge) component VIIB (Chi t 7);
Chironomus thummi thummi (midge) component VIII (Chi t 8);
Chironomus thummi thummi (midge) component X (Chi t 9);
Dolichovespula maculata (white face hornet) phospholipase (Dol m 1);
Dolichovespula maculata (white face hornet) hyaluronidase (Dol m 2);
Dolichovespula maculata (white face hornet) antigen 5 (Dol m 5);
Dolichovespula arenaria (yellow hornet) antigen 5 (Dol a 5);
Polistes annularies (wasp) phospholipase A1 (Pol a 1);
Polistes annularies (wasp) hyaluronidase (Pol a 2);
Polistes annularies (wasp) antigen 5 (Pol a 5);
Polistes dominulus (Mediterranean paper wasp) antigen (Pol d 1);
Polistes dominulus (Mediterranean paper wasp) serine protease (Pol d 4);
Polistes dominulus (Mediterranean paper wasp) antigen (Pol d 5);
Polistes exclamans (wasp) phospholipase A1 (Pol e 1);
Polistes exclamans (wasp) antigen 5 (Pol e 5);
Polistes fuscatus (wasp) antigen 5 (Pol f 5);
Polistes metricus (wasp) antigen 5 (Pol m 5);
Vespa crabo (European hornet) phospholipase (Vesp c 1);
Vespa crabo (European hornet) antigen 5 (Vesp c 5.0101);
Vespa crabo (European hornet) antigen 5 (Vesp c 5.0102);
Vespa mandarina (giant Asian hornet) antigen (Vesp m 1.01);
Vespa mandarina (giant Asian hornet) antigen (Vesp m 1.02);
Vespa mandarina (giant Asian hornet) antigen (Vesp m 5);
Vespula flavopilosa (yellowjacket) antigen 5 (Ves f 5);
Vespula germanica (yellowjacket) antigen 5 (Ves g 5);
Vespula maculifrons (yellowjacket) phospholipase A1 (Ves m 1);
Vespula maculifrons (yellowjacket) hyaluronidase (Ves m 2);
Vespula maculifrons (yellowjacket) antigen 5 (Ves m 5);
Vespula pennsylvanica (yellowjacket) (antigen 5Ves p 5);
Vespula squamosa (yellowjacket) antigen 5 (Ves s 5);
Vespula vidua (wasp) antigen (Ves vi 5);
Vespula vulgaris (yellowjacket) phospholipase A1 (Ves v 1);
Vespula vulgaris (yellowjacket) hyaluronidase (Ves v 2);
Vespula vulgaris (yellowjacket) antigen 5 (Ves v 5);
Myrmecia pilosula (Australian jumper ant) antigen (Myr p 1);
Myrmecia pilosula (Australian jumper ant) antigen (Myr p 2);
Solenopsis geminata (tropical fire ant) antigen (Sol g 2);
Solenopsis geminata (tropical fire ant) antigen (Sol g 4);
Solenopsis invicta (fire ant) antigen (Sol i 2);
Solenopsis invicta (fire ant) antigen (Sol i 3);
Solenopsis invicta (fire ant) antigen (Sol i 4);
Solenopsis saevissima (Brazilian fire ant) antigen (Sol s 2);
Gadus callarias (cod) allergen M (Gad c 1);
Salmo salar (Atlantic salmon) parvalbumin (Sal s 1);
Gallus domesticus (chicken) ovomucoid (Gal d 1);
Gallus domesticus (chicken) ovalbumin (Gal d 2);
Gallus domesticus (chicken) conalbumin; A22 (Gal d 3);
Gallus domesticus (chicken) lysozyme (Gal d 4);
Gallus domesticus (chicken) serum albumin (Gal d 5);
Metapenaeus ensis (shrimp) tropomyosin (Met e 1);
Penaeus aztecus (shrimp) tropomyosin (Pen a 1);
Penaeus indicus (shrimp) tropomyosin (Pen i 1);
Todarodes pacificus (squid) tropomyosin (Tod p 1);
Haliotis Midae (abalone) antigen (Hal m 1);
Apium graveolens (celery) Bet v 1 homologue (Api g 1);
Apium graveolens (celery) profilin (Api g 4);
Apium graveolens (celery) antigen (Api g 5);
Brassica juncea (oriental mustard) 2S albumin (Bra j 1);
Brassica rapa (turnip) prohevein-like protein (Bar r 2);
Hordeum vulgare (barley) BMAI-1 (Hor v 1);
Zea mays (maize, corn) lipid transfer protein (Zea m 14);
Corylus avellana (hazelnut) Bet v 1 homologue (Cor a 1.0401);
Malus domestica (apple) Bet v 1 homologue (Mal d 1);
Malus domestica (apple) lipid transfer protein (Mal d 3);
Pyrus communis (pear) Bet v 1 homologue (Pyr c 1);
Pyrus communis (pear) profilin (Pyr c 4);
Pyrus communis (pear) isoflavone reductase homologue (Pyr c 5);
Oryza sativa (rice) antigen (Ory s 1);
Persea americana (avocado) endochitinase (Pers a 1);
Prunus armeniaca (apricot) Bet v 1 homologue (Pru ar 1);
Prunus armeniaca (apricot) lipid transfer protein (Pru ar 3);
Prunus avium (sweet cherry) Bet v 1 homologue (Pru av 1);
Prunus avium (sweet cherry) thaumatin homologue (Pru av 2);
Prunus avium (sweet cherry) profilin (Pru av 4);
Prunus persica (peach) lipid transfer protein (Pru p 3);
Sinapis alba (yellow mustard) 2S albumin (Sin a 1);
Glycine max (soybean) HPS (Gly m 1.0101);
Glycine max (soybean) HPS (Gly m 1.0102);
Glycine max (soybean) antigen (Gly m 2);
Glycine max (soybean) profilin (Gly m 3);
Arachis hypogaea (peanut) vicilin (Ar a h 1);
Arachis hypogaea (peanut) (conglutin Ar a h 2);
Arachis hypogaea (peanut) glycinin (Ar a h 3);
Arachis hypogaea (peanut) glycinin (Ar a h 4);
Arachis hypogaea (peanut) (profilin Ar a h 5);
Arachis hypogaea (peanut) conglutin homologue (Ar a h 6);
Arachis hypogaea (peanut) conglutin homologue (Ar a h 7);
Actinidia chinensis (kiwi) cysteine protease (Act c 1);
Solanum tuberosum (potato) patatin (Sol t 1);
Bertholletia excelsa (Brazil nut) 2S albumin (Ber e 1);
Juglans regia (English walnut) 2S albumin (Jug r 1);
Juglans regia (English walnut) vicilin (Jug r 2);
Ricinus communis (castor bean) 2S albumin (Ric c 1);
Anisakis simplex (nematode) antigen (Ani s 1);
Anisakis simplex (nematode) paramyosin (Ani s 2);
Ascaris suum (worm) antigen (Asc s 1);
Aedes aegyptii (mosquito) apyrase (Aed a 1);
Aedes aegyptii (mosquito) antigen (Aed a 2);
Hevea brasiliensis (rubber) elongation factor (Hev b 1);
Hevea brasiliensis (rubber) 1,3-glucanase (Hev b 2);
Hevea brasiliensis (rubber) antigen (Hev b 3);
Hevea brasiliensis (rubber) component of microhelix protein complex (Hev b 4);
Hevea brasiliensis (rubber) antigen (Hev b 5);
Hevea brasiliensis (rubber) hevein precursor (Hev b 6.01);
Hevea brasiliensis (rubber) hevein (Hev b 6.02);
Hevea brasiliensis (rubber) C-terminal fragment antigen (Hev b 6.03);
Hevea brasiliensis (rubber) patatin homologue (Hev b 7);
Hevea brasiliensis (rubber) profilin (Hev b 8);
Hevea brasiliensis (rubber) enolase (Hev b 9);
Hevea brasiliensis (rubber) Mn-superoxide dismut (Hev b 10);
Ctenocephalides felis felis (cat flea) antigen (Cte f 1);
Homo sapiens (human autoallergen) antigen (Hom s 1);
Homo sapiens (human autoallergen) antigen (Hom s 2);
Homo sapiens (human autoallergen) antigen (Hom s 3);
Homo sapiens (human autoallergen) antigen (Hom s 4); and
Homo sapiens (human autoallergen) antigen (Hom s 5),

the expressed protein allergen being encapsulated within the dead E. coli; and
a pharmaceutically acceptable carrier,

wherein the administered dead E. coli are taken up by antigen presenting cells in the subject, the recombinant version of the allergen protein is released and processed inside the antigen presenting cells, and the processed protein is displayed on the antigen presenting cell surface,
so that one or more symptoms of the subject's allergy to the protein allergen is reduced or decreased.

3. A method of treating allergy in a subject susceptible to an allergic response to a protein allergen, the method comprising a step of:
administering to a subject, via the subject's rectum, a composition comprising:
dead E. coli that has expressed non-secreted version of a protein allergen selected from the group consisting of:

Ambrosia artemisiifolia (short ragweed) antigen E (Amb a 1);
Ambrosia artemisiifolia (short ragweed) antigen K (Amb a 2);
Ambrosia artemisiifolia (short ragweed) Ra3 antigen (Amb a 3);
Ambrosia artemisiifolia (short ragweed) Ra5 antigen (Amb a 5);
Ambrosia artemisiifolia (short ragweed) Ra6 antigen (Amb a 6);
Ambrosia artemisiifolia (short ragweed) Ra7 antigen (Amb a 7);
Ambrosia trifida (giant ragweed) Ra5G antigen (Amb t 5);
Artemisia vulgaris (mugwort) antigen (Art v 1);
Artemisia vulgaris (mugwort) antigen (Art v 2);
Helianthus annuus (sunflower) antigen (Hel a 1);
Helianthus annuus (sunflower) profilin (Hel a 2);
Mercurialis annua (annual mercury) profilin (Mer a 1);
Cynodon dactylon (Bermuda grass) antigen (Cyn d 1);
Cynodon dactylon (Bermuda grass) antigen (Cyn d 7);
Cynodon dactylon (Bermuda grass) profilin (Cyn d 12);
Dactylis glomerata (orchard grass) AgDg1 antigen (Dac g 1);
Dactylis glomerata (orchard grass) antigen (Dac g 2);
Dactylis glomerata (orchard grass) antigen (Dac g 3);
Dactylis glomerata (orchard grass) antigen (Dac g 5);
Holcus lanatus (velvet grass) antigen (Hol l 1);
Lolium perenne (rye grass) group I antigen (Lol p 1);
Lolium perenne (rye grass) group II antigen (Lol p 2);
Lolium perenne (rye grass) group III antigen (Lol p 3);
Lolium perenne (rye grass) group IX antigen (Lol p 5);
Lolium perenne (rye grass) antigen (Lol p Ib);
Lolium perenne (rye grass) trypsin (Lol p 11);
Phalaris aquatica (canary grass) antigen (Pha a 1);
Phleum pratense (timothy grass) antigen (Phl p 1);
Phleum pratense (timothy grass) antigen (Phl p 2);
Phleum pratense (timothy grass) antigen (Phl p 4);
Phleum pratense (timothy grass) antigen Ag 25 (Phl p 5);
Phleum pratense (timothy grass) antigen (Phl p 6);
Phleum pratense (timothy grass) profilin (Phl p 12);
Phleum pratense (timothy grass) polygalacturonase (Phl p 13);
Poa pratensis (Kentucky blue grass) group I antigen (Poa p 1);
Poa pratensis (Kentucky blue grass) antigen (Poa p 5);
Sorghum halepense (Johnson grass) antigen (Sor h 1);
Alnus glutinosa (alder) antigen (Aln g 1);
Betula verrucosa (birch) antigen (Bet v 1);
Betula verrucosa (birch) profilin (Bet v 2);
Betula verrucosa (birch) antigen (Bet v 3);
Betula verrucosa (birch) antigen (Bet v 4);
Betula verrucosa (birch) isoflavone reductase homologue (Bet v 5);
Betula verrucosa (birch) cyclophilin (Bet v 7);
Carpinus betulus (hornbeam) antigen (Car b 1);
Castanea sativa (chestnut) Bet v 1 homologue (Cas s 1);
Castanea sativa (chestnut) chitinase (Cas s 5);
Corylus avelana (hazel) antigen (Cor a 1);
Quercus alba (white oak) antigen (Que a 1);
Cryptomeria japonica (sugi) antigen (Cry j 1);
Cryptomeria japonica (sugi) antigen (Cry j 2);
Juniperus ashei (mountain cedar) antigen (Jun a 1);
Juniperus ashei (mountain cedar) antigen (Jun a 3);
Juniperus oxycedrus (prickly juniper) calmodulin-like antigen (Jun o 2);
Juniperus sabinoides (mountain cedar) antigen (Jun s 1);
Juniperus virginiana (eastern red cedar) antigen (Jun v 1);
Fraxinus excelsior (ash) antigen (Fra e 1);
Ligustrum vulgare (privet) antigen (Lig v 1);
Olea europea (olive) antigen (Ole e 1);
Olea europea (olive) profilin (Ole e 2);
Olea europea (olive) antigen (Ole e 3);
Olea europea (olive) antigen (Ole e 4);
Olea europea (olive) superoxide dismutase (Ole e 5);
Olea europea (olive) antigen (Ole e 6);
Syringa vulgaris (lilac) antigen (Syr v 1);
Acarus siro (mite) fatty acid-binding protein (Aca s 13);
Blomia tropicalis (mite) antigen (Blo t 5);
Blomia tropicalis (mite) Bt11a antigen (Blo t 12);
Blomia tropicalis (mite) Bt6 fatty acid-binding protein (Blo t);
Dermatophagoides pteronyssinus (mite) antigen P1 (Der p 1);
Dermatophagoides pteronyssinus (mite) antigen (Der p 2);
Dermatophagoides pteronyssinus (mite) trypsin (Der p 3);
Dermatophagoides pteronyssinus (mite) amylase (Der p 4);
Dermatophagoides pteronyssinus (mite) antigen (Der p 5);
Dermatophagoides pteronyssinus (mite) chymotrypsin (Der p 6);
Dermatophagoides pteronyssinus (mite) antigen (Der p 7);
Dermatophagoides pteronyssinus (mite) glutathione transferase (Der p 8);
Dermatophagoides pteronyssinus (mite) collagenolytic serine prot. (Der p 9);
Dermatophagoides pteronyssinus (mite) tropomyosin (Der p 10);
Dermatophagoides pteronyssinus (mite) apolipophorin like p (Der p 14);
Dermatophagoides microceras (mite) antigen (Der m 1);
Dermatophagoides farinae (mite) antigen (Der f 1);
Dermatophagoides farinae (mite) antigen (Der f 2);
Dermatophagoides farinae (mite) antigen (Der f 3);
Dermatophagoides farinae (mite) tropomyosin (Der f 10);
Dermatophagoides farinae (mite) paramyosin (Der f 11);
Dermatophagoides farinae (mite) Mag 3, apolipophorin (Der f 14);
Euroglyphus maynei (mite) apolipophorin (Eur m 14);
Lepidoglyphus destructor (storage mite) antigen (Lep d 2.0101);
Lepidoglyphus destructor (storage mite) antigen (Lep d 2.0102);
Bos domesticus (cow) Ag3, lipocalin (Bos d 2);
Bos domesticus (cow) alpha-lactalbumin (Bos d 4);
Bos domesticus (cow) beta-lactalbumin (Bos d 5);
Bos domesticus (cow) serum albumin (Bos d 6);
Bos domesticus (cow) immunoglobulin (Bos d 7);
Bos domesticus (cow) casein (Bos d 8);
Canis familiaris (dog) antigen (Can f 1);
Canis familiaris (dog) antigen (Can f 2);
Canis familiaris (dog) albumin (Can f ?);
Equus caballus (horse) lipocalin (Equ c 1);
Equus caballus (horse) lipocalin (Equ c 2);
Felis domesticus (cat) cat-1 antigen (Fel d 1);
Mus musculus (mouse) MUP antigen (Mus m 1);
Rattus norvegius (rat) antigen (Rat n 1);
Alternaria alternata (fungus) antigen (Alt a 1);
Alternaria alternata (fungus) antigen (Alt a 2);
Alternaria alternata (fungus) heat shock protein (Alt a 3);
Alternaria alternata (fungus) ribosomal protein (Alt a 6);
Alternaria alternata (fungus) YCP4 protein (Alt a 7);
Alternaria alternata (fungus) aldehyde dehydrogenase (Alt a 10);
Alternaria alternata (fungus) enloase (Alt a 11);
Alternaria alternata (fungus) acid ribosomal protein P1 (Alt a 12);
Cladosporium herbarum (fungus) antigen (Cla h 1);
Cladosporium herbarum (fungus) antigen (Cla h 2);
Cladosporium herbarum (fungus) aldehyde dehydrogenase (Cla h 3);
Cladosporium herbarum (fungus) ribosomal protein);
Cladosporium herbarum (fungus) YCP4 protein (Cla h 5);
Cladosporium herbarum (fungus) enolase (Cla h 6);
Cladosporium herbarum (fungus) acid ribosomal protein P1 (Cla h 12);
Aspergillus flavus (fungus) alkaline serine proteinase (Asp fl 13);
Aspergillus Fumigatus (fungus) antigen (Asp f 1);
Aspergillus Fumigatus (fungus) antigen (Asp f 2);
Aspergillus Fumigatus (fungus) peroxisomal protein (Asp f 3);
Aspergillus Fumigatus (fungus) antigen (Asp f 4);
Aspergillus Fumigatus (fungus) metalloprotease (Asp f 5);
Aspergillus Fumigatus (fungus) Mn superoxide dismutase (Asp f 6);
Aspergillus Fumigatus (fungus) antigen (Asp f 7);
Aspergillus Fumigatus (fungus) ribosomal protein P2 (Asp f 8);
Aspergillus Fumigatus (fungus) antigen (Asp f 9);
Aspergillus Fumigatus (fungus) aspartis protease (Asp f 10);
Aspergillus Fumigatus (fungus) peptidyl-prolyl isomerase (Asp f 11);
Aspergillus Fumigatus (fungus) heat shock protein P70 (Asp f 12);
Aspergillus Fumigatus (fungus) alkaline serine proteinase (Asp f 13);
Aspergillus Fumigatus (fungus) antigen (Asp f 15);
Aspergillus Fumigatus (fungus) antigen (Asp f 16);
Aspergillus Fumigatus (fungus) antigen (Asp f 17);
Aspergillus Fumigatus (fungus) vacuolar serine (Asp f 18);
Aspergillus niger (fungus) beta-xylosidase (Asp n 14);
Aspergillus niger (fungus) antigen (Asp n 18);
Aspergillus niger (fungus) vacuolar serine proteinase;
Aspergillus oryzae (fungus) TAKA-amylase A (Asp o 2);
Aspergillus oryzae (fungus) alkaline serine proteinase (Asp o 13);
Penicillium brevicompactum (fungus) alkaline serine proteinase (Pen b 13);
Penicillium citrinum (fungus) heat shock protein P70 (Pen c 1);
Penicillium citrinum (fungus) peroxisomal membrane protein (Pen c 3);
Penicillium citrinum (fungus) alkaline serine proteinase (Pen c 13);
Penicillium notatum (fungus) N-acetyl glucosaminidase (Pen n 1);
Penicillium notatum (fungus) alkaline serine proteinase (Pen n 13);
Penicillium notatum (fungus) vacuolar serine proteinase (Pen n 18);
Penicillium oxalicum (fungus) vacuolar serine proteinase (Pen o 18);
Trichophyton rubrum (fungus) antigen (Tri r 2);
Trichophyton rubrum (fungus) serine protease (Tri r 4);
Trichophyton tonsurans (fungus) antigen (Tri t 1);
Trichophyton tonsurans (fungus) serine protease (Tri t 4);
Candida albicans (fungus) antigen (Cand a 1);
Candida boidinii (fungus) antigen (Cand b 2);
Malassezia furfur (fungus) antigen (Mal f 1);
Malassezia furfur (fungus) MF1 peroxisomal membrane protein (Mal f 2);
Malassezia furfur (fungus) MF2 peroxisomal membrane protein (Mal f 3);
Malassezia furfur (fungus) antigen (Mal f 4);
Malassezia furfur (fungus) antigen (Mal f 5);
Malassezia furfur (fungus) cyclophilin homologue (Mal f 6);
Psilocybe cubensis (fungus) antigen (Psi c 1);
Psilocybe cubensis (fungus) cyclophilin (Psi c 2);
Coprinus comatus (shaggy cap) antigen (Cop c 1);
Coprinus comatus (shaggy cap) antigen (Cop c 2);
Coprinus comatus (shaggy cap) antigen (Cop c 3);
Coprinus comatus (shaggy cap) antigen (Cop c 5);
Coprinus comatus (shaggy cap) antigen (Cop c 7);
Aedes aegyptii (mosquito) apyrase (Aed a 1);
Aedes aegyptii (mosquito) antigen (Aed a 2);
Apis mellifera (honey bee) phospholipase A2 (Api m 1);
Apis mellifera (honey bee) hyaluronidase (Api m 2);
Apis mellifera (honey bee) melittin (Api m 4);
Apis mellifera (honey bee) antigen (Api m 6);
Bombus pennsylvanicus (bumble bee) phospholipase (Bom p 1);
Bombus pennsylvanicus (bumble bee) protease (Bom p 4);
Blattella germanica (German cockroach) Bd90k (Bla g 1);
Blattella germanica (German cockroach) aspartic protease (Bla g 2);
Blattella germanica (German cockroach) calycin (Bla g 4);
Blattella germanica (German cockroach) glutathione transferase (Bla g 5);
Blattella germanica (German cockroach) troponin C (Bla g 6);
Periplaneta americana (American cockroach) Cr-PII (Per a 1);
Periplaneta americana (American cockroach) Cr-PI (Per a 3);
Periplaneta americana (American cockroach) tropomyosin (Per a 7);
Chironomus thummi thummi (midge) hemoglobin (Chi t 1-9);
Chironomus thummi thummi (midge) component III (Chi t 1.01);
Chironomus thummi thummi (midge) component IV (Chi t 1.02);
Chironomus thummi thummi (midge) component I (Chi t 2.0101);
Chironomus thummi thummi (midge) component IA (Chi t 2.0102);
Chironomus thummi thummi (midge) component II-beta (Chi t 3);
Chironomus thummi thummi (midge) component IIIA (Chi t 4);
Chironomus thummi thummi (midge) component VI (Chi t 5);
Chironomus thummi thummi (midge) component VIIA (Chi t 6.01);
Chironomus thummi thummi (midge) component IX (Chi t 6.02);
Chironomus thummi thummi (midge) component VIIB (Chi t 7);
Chironomus thummi thummi (midge) component VIII (Chi t 8);
Chironomus thummi thummi (midge) component X (Chi t 9);
Dolichovespula maculata (white face hornet) phospholipase (Dol m 1);
Dolichovespula maculata (white face hornet) hyaluronidase (Dol m 2);
Dolichovespula maculata (white face hornet) antigen 5 (Dol m 5);
Dolichovespula arenaria (yellow hornet) antigen 5 (Dol a 5);
Polistes annularies (wasp) phospholipase A1 (Pol a 1);
Polistes annularies (wasp) hyaluronidase (Pol a 2);
Polistes annularies (wasp) antigen 5 (Pol a 5);
Polistes dominulus (Mediterranean paper wasp) antigen (Pol d 1);
Polistes dominulus (Mediterranean paper wasp) serine protease (Pol d 4);
Polistes dominulus (Mediterranean paper wasp) antigen (Pol d 5);
Polistes exclamans (wasp) phospholipase A1 (Pol e 1);
Polistes exclamans (wasp) antigen 5 (Pol e 5);
Polistes fuscatus (wasp) antigen 5 (Pol f 5);
Polistes metricus (wasp) antigen 5 (Pol m 5);
Vespa crabo (European hornet) phospholipase (Vesp c 1);
Vespa crabo (European hornet) antigen 5 (Vesp c 5.0101);
Vespa crabo (European hornet) antigen 5 (Vesp c 5.0102);
Vespa mandarina (giant Asian hornet) antigen (Vesp m 1.01);
Vespa mandarina (giant Asian hornet) antigen (Vesp m 1.02);
Vespa mandarina (giant Asian hornet) antigen (Vesp m 5);
Vespula flavopilosa (yellowjacket) antigen 5 (Ves f 5);
Vespula germanica (yellowjacket) antigen 5 (Ves g 5);
Vespula maculifrons (yellowjacket) phospholipase A1 (Ves m 1);
Vespula maculifrons (yellowjacket) hyaluronidase (Ves m 2);
Vespula maculifrons (yellowjacket) antigen 5 (Ves m 5);
Vespula pennsylvanica (yellowjacket) (antigen 5Ves p 5);
Vespula squamosa (yellowjacket) antigen 5 (Ves s 5);
Vespula vidua (wasp) antigen (Ves vi 5);
Vespula vulgaris (yellowjacket) phospholipase A1 (Ves v 1);
Vespula vulgaris (yellowjacket) hyaluronidase (Ves v 2);
Vespula vulgaris (yellowjacket) antigen 5 (Ves v 5);
Myrmecia pilosula (Australian jumper ant) antigen (Myr p 1);
Myrmecia pilosula (Australian jumper ant) antigen (Myr p 2);
Solenopsis geminata (tropical fire ant) antigen (Sol g 2);
Solenopsis geminata (tropical fire ant) antigen (Sol g 4);
Solenopsis invicta (fire ant) antigen (Sol i 2);
Solenopsis invicta (fire ant) antigen (Sol i 3);
Solenopsis invicta (fire ant) antigen (Sol i 4);
Solenopsis saevissima (Brazilian fire ant) antigen (Sol s 2);
Gadus callarias (cod) allergen M (Gad c 1);
Salmo salar (Atlantic salmon) parvalbumin (Sal s 1);
Gallus domesticus (chicken) ovomucoid (Gal d 1);
Gallus domesticus (chicken) ovalbumin (Gal d 2);
Gallus domesticus (chicken) conalbumin; A22 (Gal d 3);
Gallus domesticus (chicken) lysozyme (Gal d 4);
Gallus domesticus (chicken) serum albumin (Gal d 5);
Metapenaeus ensis (shrimp) tropomyosin (Met e 1);
Penaeus aztecus (shrimp) tropomyosin (Pen a 1);
Penaeus indicus (shrimp) tropomyosin (Pen i 1);
Todarodes pacificus (squid) tropomyosin (Tod p 1);
Haliotis Midae (abalone) antigen (Hal m 1);
Apium graveolens (celery) Bet v 1 homologue (Api g 1);
Apium graveolens (celery) profilin (Api g 4);
Apium graveolens (celery) antigen (Api g 5);
Brassica juncea (oriental mustard) 2S albumin (Bra j 1);
Brassica rapa (turnip) prohevein-like protein (Bar r 2);
Hordeum vulgare (barley) BMAI-1 (Hor v 1);
Zea mays (maize, corn) lipid transfer protein (Zea m 14);
Corylus avellana (hazelnut) Bet v 1 homologue (Cor a 1.0401);
Malus domestica (apple) Bet v 1 homologue (Mal d 1);
Malus domestica (apple) lipid transfer protein (Mal d 3);
Pyrus communis (pear) Bet v 1 homologue (Pyr c 1);
Pyrus communis (pear) profilin (Pyr c 4);
Pyrus communis (pear) isoflavone reductase homologue (Pyr c 5);
Oryza sativa (rice) antigen (Ory s 1);
Persea americana (avocado) endochitinase (Pers a 1);
Prunus armeniaca (apricot) Bet v 1 homologue (Pru ar 1);
Prunus armeniaca (apricot) lipid transfer protein (Pru ar 3);
Prunus avium (sweet cherry) Bet v 1 homologue (Pru av 1);
Prunus avium (sweet cherry) thaumatin homologue (Pru av 2);
Prunus avium (sweet cherry) profilin (Pru av 4);
Prunus persica (peach) lipid transfer protein (Pru p 3);
Sinapis alba (yellow mustard) 2S albumin (Sin a 1);
Glycine max (soybean) HPS (Gly m 1.0101);
Glycine max (soybean) HPS (Gly m 1.0102);
Glycine max (soybean) antigen (Gly m 2);
Glycine max (soybean) profilin (Gly m 3);
Arachis hypogaea (peanut) vicilin (Ar a h 1);
Arachis hypogaea (peanut) (conglutin Ar a h 2);
Arachis hypogaea (peanut) glycinin (Ar a h 3);
Arachis hypogaea (peanut) glycinin (Ar a h 4);
Arachis hypogaea (peanut) (profilin Ar a h 5);
Arachis hypogaea (peanut) conglutin homologue (Ar a h 6);
Arachis hypogaea (peanut) conglutin homologue (Ar a h 7);
Actinidia chinensis (kiwi) cysteine protease (Act c 1);
Solanum tuberosum (potato) patatin (Sol t 1);
Bertholletia excelsa (Brazil nut) 2S albumin (Ber e 1);
Juglans regia (English walnut) 2S albumin (Jug r 1);
Juglans regia (English walnut) vicilin (Jug r 2);
Ricinus communis (castor bean) 2S albumin (Ric c 1);
Anisakis simplex (nematode) antigen (Ani s 1);
Anisakis simplex (nematode) paramyosin (Ani s 2);
Ascaris suum (worm) antigen (Asc s 1);
Aedes aegyptii (mosquito) apyrase (Aed a 1);
Aedes aegyptii (mosquito) antigen (Aed a 2);
Hevea brasiliensis (rubber) elongation factor (Hev b 1);
Hevea brasiliensis (rubber) 1,3-glucanase (Hev b 2);
Hevea brasiliensis (rubber) antigen (Hev b 3);
Hevea brasiliensis (rubber) component of microhelix protein complex (Hev b 4);
Hevea brasiliensis (rubber) antigen (Hev b 5);
Hevea brasiliensis (rubber) hevein precursor (Hev b 6.01);
Hevea brasiliensis (rubber) hevein (Hev b 6.02);
Hevea brasiliensis (rubber) C-terminal fragment antigen (Hev b 6.03);
Hevea brasiliensis (rubber) patatin homologue (Hev b 7);
Hevea brasiliensis (rubber) profilin (Hev b 8);
Hevea brasiliensis (rubber) enolase (Hev b 9);
Hevea brasiliensis (rubber) Mn-superoxide dismut (Hev b 10);
Ctenocephalides felis felis (cat flea) antigen (Cte f 1);
Homo sapiens (human autoallergen) antigen (Hom s 1);
Homo sapiens (human autoallergen) antigen (Hom s 2);
Homo sapiens (human autoallergen) antigen (Hom s 3);
Homo sapiens (human autoallergen) antigen (Hom s 4); and
Homo sapiens (human autoallergen) antigen (Hom s 5),

the expressed protein allergen being encapsulated within the dead E. coli; and
a pharmaceutically acceptable carrier,

so that one or more symptoms of the subject's allergy to the protein allergen is reduced or decreased.

4. A method of treating peanut allergy in a subject susceptible to an allergic response to a protein allergen, the method comprising a step of:
administering to a subject, via the subject's rectum, a composition comprising:
dead E. coli that has expressed a protein allergen selected from the group consisting of peanut allergens Ara h 1, Ara h 2, and Ara h 3, the expressed protein allergen being encapsulated within the dead E. coli; and
a pharmaceutically acceptable carrier,


wherein the administered dead E. coli are taken up by antigen presenting cells in the subject, the recombinant version of the peanut allergen is released and processed inside the antigen presenting cells, and the processed allergen is displayed on the antigen presenting cell surface,
so that one or more symptoms of the subject's allergy to the protein allergen is reduced or decreased.

5. A method of treating peanut allergy in a subject susceptible to an allergic response to a protein allergen, the method comprising a step of:
administering to a subject, via the subject's rectum, a composition comprising:
dead E. coli that has expressed a non-secreted version of a protein allergen selected from the group consisting of peanut allergens Ara h 1, Ara h 2, and Ara h 3, the expressed protein allergen being encapsulated within the dead E. coli; and
a pharmaceutically acceptable carrier,


so that one or more symptoms of the subject's allergy to the protein allergen is reduced or decreased.

6. The method of any one of claims 1, 2 and 3, wherein the subject is a mammal.

7. The method of any one of claims 1, 2 and 3, wherein the subject is a human.

8. The method of any one of claims 1, 2 and 3, wherein in the step of administering, the composition is delivered in the form of a suppository.

9. The method of any one of claims 1, 2 and 3, wherein in the step of administering, the composition is delivered in the form of an enema.

10. The method of any one of claims 1, 2 and 3, wherein in the step of administering, the composition is delivered using a rectal catheter.

11. The method of any one of claims 1, 2 and 3, further comprising a step of identifying a subject susceptible to an allergic response to a protein allergen, wherein the subject is identified as an individual with an elevated level of IgE antibodies that bind with the protein allergen, and wherein the step of administering comprises administering to the identified subject.

12. The method of any one of claims 1, 2 and 3, further comprising a step of identifying a subject susceptible to an allergic response to a protein allergen, wherein the subject is identified as an individual with a positive skin prick test to the protein allergen, and wherein the step of administering comprises administering to the identified subject.

13. The method of any one of claims 1, 2 and 3, further comprising a step of identifying a subject susceptible to an allergic response to a protein allergen, wherein the subject is identified as an individual having a characteristic selected from the group consisting of a prior display of allergic symptoms when exposed to the protein allergen and a familial relationship with an individual who previously displayed allergic symptoms when exposed to the protein allergen, and wherein the step of administering comprises administering to the identified subject.

14. A method of treating peanut allergy in a subject susceptible to an allergic response to a protein allergen selected from the group consisting of peanut allergens Ara h 1, Ara h 2, and Ara h 3, the method comprising:
administering to a subject, via the subject's rectum, a composition comprising:
dead E. coli cells having expressed a peanut allergen selected from the group consisting of Ara h1 of SEQ ID NO: 2, Ara h2 of SEQ ID NO: 4 and Ara h3 of SEQ ID NO: 6 so that the expressed allergen is encapsulated within the dead E. coli cells and is not exposed to the subject's IgE antibodies during administration; and
a pharmaceutically acceptable carrier,


so that one or more symptoms of the subject's allergy to the protein allergen is reduced or decreased.

15. The method of any one of claims 4, 5 and 14, wherein the protein allergen is a modified peanut allergen protein whose amino acid sequence differs from that of an intact wild-type peanut allergen protein in that at least one IgE epitope has a substitution in the modified peanut allergen protein such that the modified peanut allergen protein has a reduced ability to bind to or cross-link IgE as compared with the unmodified peanut allergen protein, the at least one IgE epitope being one that is recognized when the unmodified peanut allergen protein is contacted with serum IgE from an individual that is allergic to the unmodified peanut allergen protein, wherein:
when the wild-type peanut protein has the amino acid sequence of SEQ ID NO: 2, the substitution is in an IgE epitope selected from the group consisting of:
an epitope found between amino acids 25 and 34 of SEQ ID NO: 2;
an epitope found between amino acids 48 and 57 of SEQ ID NO: 2;
an epitope found between amino acids 65 and 74 of SEQ ID NO: 2;
an epitope found between amino acids 89 and 98 of SEQ ID NO: 2;
an epitope found between amino acids 97 and 106 of SEQ ID NO: 2;
an epitope found between amino acids 107 and 116 of SEQ ID NO: 2;
an epitope found between amino acids 123 and 132 of SEQ ID NO: 2;
an epitope found between amino acids 134 and 143 of SEQ ID NO: 2;
an epitope found between amino acids 294 and 303 of SEQ ID NO: 2;
an epitope found between amino acids 311 and 320 of SEQ ID NO: 2;
an epitope found between amino acids 325 and 334 of SEQ ID NO: 2;
an epitope found between amino acids 344 and 353 of SEQ ID NO: 2;
an epitope found between amino acids 393 and 402 of SEQ ID NO: 2;
an epitope found between amino acids 409 and 418 of SEQ ID NO: 2;
an epitope found between amino acids 461 and 470 of SEQ ID NO: 2;
an epitope found between amino acids 498 and 507 of SEQ ID NO: 2;
an epitope found between amino acids 525 and 534 of SEQ ID NO: 2;
an epitope found between amino acids 539 and 548 of SEQ ID NO: 2;
an epitope found between amino acids 551 and 560 of SEQ ID NO: 2;
an epitope found between amino acids 559 and 568 of SEQ ID NO: 2;
an epitope found between amino acids 578 and 587 of SEQ ID NO: 2;
an epitope found between amino acids 597 and 606 of SEQ ID NO: 2;
and a combination thereof; and

when the wild-type peanut protein has the amino acid sequence of SEQ ID NO: 4, the substitution is in an IgE epitope selected from the group consisting of:
an epitope found between amino acids 15 and 24 of SEQ ID NO: 4;
an epitope found between amino acids 21 and 30 of SEQ ID NO: 4;
an epitope found between amino acids 27 and 36 of SEQ ID NO: 4;
an epitope found between amino acids 39 and 48 of SEQ ID NO: 4;
an epitope found between amino acids 49 and 58 of SEQ ID NO: 4;
an epitope found between amino acids 57 and 66 of SEQ ID NO: 4;
an epitope found between amino acids 65 and 74 of SEQ ID NO: 4;
an epitope found between amino acids 115 and 124 of SEQ ID NO: 4;
an epitope found between amino acids 127 and 136 of SEQ ID NO: 4;
an epitope found between amino acids 143 and 152 of SEQ ID NO: 4;
and a combination thereof; and

when the wild-type peanut protein has the amino acid sequence of SEQ ID NO: 6, the substitution is in an IgE epitope selected from the group consisting of:
an epitope found between amino acids 33 and 47 of SEQ ID NO: 6;
an epitope found between amino acids 240 and 254 of SEQ ID NO: 6;
an epitope found between amino acids 279 and 293 of SEQ ID NO: 6;
an epitope found between amino acids 303 and 317 of SEQ ID NO: 6;
and a combination thereof.

16. The method of any one of claims 4, 5 and 14, wherein the subject is a mammal.

17. The method of any one of claims 4, 5 and 14, wherein the subject is a human.

18. The method of any one of claims 4, 5 and 14, wherein in the step of administering, the composition is delivered in the form of a suppository.

19. The method of any one of claims 4, 5 and 14, wherein in the step of administering, the composition is delivered in the form of an enema.

20. The method of any one of claims 4, 5 and 14, wherein in the step of administering, the composition is delivered using a rectal catheter.

21. The method of any one of claims 4, 5 and 14, further comprising a step of identifying a subject susceptible to an allergic response to a protein allergen, wherein the subject is identified as an individual with an elevated level of IgE antibodies that bind with the protein allergen, and wherein the step of administering comprises administering to the identified subject.

22. The method of any one of claims 4, 5 and 14, further comprising a step of identifying a subject susceptible to an allergic response to a protein allergen, wherein the subject is identified as an individual with a positive skin prick test to the protein allergen, and wherein the step of administering comprises administering to the identified subject.

23. The method of any one of claims 4, 5 and 14, further comprising a step of identifying a subject susceptible to an allergic response to a protein allergen, wherein the subject is identified as an individual having a characteristic selected from the group consisting of a prior display of allergic symptoms when exposed to the protein allergen and a familial relationship with an individual who previously displayed allergic symptoms when exposed to the protein allergen, and wherein the step of administering comprises administering to the identified subject.

24. The method of any one of claims 4, 5 and 14, wherein the protein allergen has an amino acid sequence that shows 100% identity with that of a wild-type allergen as it occurs in nature.