WO2001034186A1 - Compositions et procedes permettant de modifier une reponse immunitaire contre la tropomyosine - Google Patents

Compositions et procedes permettant de modifier une reponse immunitaire contre la tropomyosine Download PDF

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WO2001034186A1
WO2001034186A1 PCT/US2000/030968 US0030968W WO0134186A1 WO 2001034186 A1 WO2001034186 A1 WO 2001034186A1 US 0030968 W US0030968 W US 0030968W WO 0134186 A1 WO0134186 A1 WO 0134186A1
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tropomyosin
crustacea
amino acid
ige
modified
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PCT/US2000/030968
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English (en)
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Samuel B. Lehrer
Gerald Reese
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Lehrer Samuel B
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Priority to AU15970/01A priority Critical patent/AU1597001A/en
Priority to CA002391421A priority patent/CA2391421A1/fr
Publication of WO2001034186A1 publication Critical patent/WO2001034186A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • the present invention relates to compositions and methods for modifying an immune response against tropomyosin and related antigens.
  • the invention provides vaccines including modified tropomyosin that reduce or eliminate an unwanted immune response against tropomyosin. Also provided are modified tropomyosin molecules with modified antibody binding sites (epitopes) that significantly reduce or eliminate potential to engage the immune system.
  • the invention also provides transgenic animals, particularly Crustacea, that included at least one of the modified tropomyosin molecules described herein. The invention has wide applicability including reducing harmful immune responses to Crustacea, arthropods, and other animals.
  • shellfish crustaceans and mollusks
  • shellfish crustaceans and mollusks
  • the only major allergan reported in shrimp is the muscle protein tropomyosin. At least 80% shrimp- allergic subjects react to tropomyosin and it binds approximately 85% of the shrimp specific IgE from shrimp-allergic subjects; all other shrimp allergans bind IgE from less than 25% of the shrimp-allergic subjects.
  • tropomyosin is an important allergan in other crustaceans such as lobsterP ⁇ nuftrus stimpsoni and Homarus americanus (Pan s 1, Horn a 1) , crab Charyabdis fe ⁇ atus (Cha f 1), mollusk such as squid Todareus pacificus (Tod p i), snail Turbo cornutus (Tur c 1) and oyster Crassostrea gigas (Cra g 1) and in other invertebrates such as house dust mite Dermatophagoides farinae (Der f 10) and D.
  • crustaceans such as lobsterP ⁇ nuftrus stimpsoni and Homarus americanus (Pan s 1, Horn a 1) , crab Charyabdis fe ⁇ atus (Cha f 1), mollusk such as squid Todareus pacificus (Tod p i), snail Turbo cornutus (Tur c 1) and oyster
  • tropomyosin belongs to a family of proteins present in all eukaryotic cells, where it is associated with the thin filament in muscle, and microfilaments in many non-muscle cells. Together with actin and myoan, tropomyosin plays a role in the contractile activities of these cells, as well as in the regulation of cell morphology and motility.
  • Tropomyosin is thought to be present in phylogenetically unrelated vertebrate and invertebrate species, with several tropomyosin isoforms being found in muscle (skeletal, cardiac and smooth), and non-muscle cells such as those in brain, fibroblasts and platelets. Even though the degree of sequence identity and functional similarity is reported to be very high among tropomyosins, vertebrate tropomyosins are generally considered to be non aller genie.
  • Tropomyosins have attracted substantial research interest.
  • the proteins are coiled-coil dimers made up of two parallel ⁇ -helical tropomyosin molecules that are wound around each other.
  • the tropomyosin monomer contains a heptad repeat, (abcdefg) ⁇ in which generally large hydrophobic nonpolar residues occur at positions a and d, while positions b, c, e, f and g are usually occupiedby polar or ionic amino acids.
  • the interaction between two alpha-helices in a coiled-coil involve these hydrophobic residues in position a and d.
  • Tests such as skin prick test (SPT) have been used to gauge risk of allergy to tropomyosin and other molecules.
  • the invention generally relates to compositions and methods for modifying an immune response against tropomyosin. As discussed below, the invention has many important uses including reducing or eliminating harmful immune responses against the tropomyosin of animals such as Crustacea and arthropods. Also provided are transgenic animals and especially transgenic Crustacea that include at least one recombinant tropomyosin specifically modified to reduce or eliminate the unwanted immune responses.
  • tropomyosin sites that engage the vertebrate immune system and elicit harmful immune responses. More specifically, we have identified the Pen a 1 (tropomyosin) epitopes that elicit IgE- mediated immune responses. As related below, we have found about 15 - 20 IgE binding regions in the tropomyosin molecule. We have found one epitope for region 1, one epitope for region 2, two epitopes for region 3, one epitope for region 4 and three epitopes for region 5.
  • the invention provides vaccines that include at least one modified tropomyosin that reduces or eliminates an unwanted immune response against tropomyosin.
  • a modified tropomyosin can, in one embodiment, include at least one amino acid substitution in at least one of the foregoing regions one to five of tropomyosin.
  • Other vaccines according to the invention will include at least a fragment of tropomyosin and may also include at least one of the modified tropomyosins described herein.
  • the vaccine will include at least one modified amino acid that reduces binding, preferably specific, between Crustacea tropomyosin and an IgE antibody of interest by at least about 45% as determined by a standard IgE antibody test.
  • suitably modified amino acid sequences will include at least one epitope with at least one amino acid substitution that reduces the binding according to the test. Examples of such preferred amino acid substitutions include those which remove at least one of a non-polar aliphatic, polar unchaiged, aromatic, positively charged or negatively charged group from the amino acid.
  • another preferred vaccine includes Crustacea tropomyosin or an IgE antibody eliciting fragment thereof.
  • exposure to the tropomyosin is thought to condition the immune system against significantly engaging the molecule as part of a harmful response.
  • modified tropomyosin molecules such as peptides with modified antibody binding sites (epitopes) that significantly reduce or eliminate potential to engage the immune system.
  • the invention also provides transgenic animals, particularly Crustacea, that include at least one of the modified tropomyosin molecules described herein.
  • the invention has wide applicability including reducing harmful immune responses to Crustacea, arthropods, and other animals.
  • Tropomyosin is an essential muscle protein that is present in all animal species and is highly conserved.
  • invertebrate tropomyosins have amino acid sequence homology with vertebrate tropomyosins such as those present in non- allergenic foods such as beef, pork and chicken.
  • homologous region of non-allergenic tropomyosin as a template to alter the allergenic epitopes of shrimp tropomyosin to render them inactive. The aim was to make minimal changes in the allergenic epitopes that would induce maximal reduction of tropomyosin-specific IgE binding.
  • the invention provides a series of 46 overlapping peptides that span the entire 284 amino acid residue of Pen a 1 tropomyosin protein. Each of these peptides were used to identify the IgE binding epitopes of Pen a 1. As used herein, IgE binding epitopes are defined as any sequence of Pen a 1 that binds Pen a 1 -specific IgE of shrimp allergic subjects.
  • the invention provides for peptides that have reduced or totally lack binding ability to Pen a 1-specific IgE.
  • Peptides were generated that had one or more amino acid substitutions based on, but not limited to, sequence comparisons with non-allergenic tropomyosins.
  • modified Pen a 1 peptides or Pen a 1 molecules with reduced or abolished IgE binding capacity are defined as any Pen a 1 molecule or peptide that contain one or more amino acid substitutions that reduce or abolish IgE binding of the peptide or Pen a 1 molecule.
  • the present invention provides for oral and/ or immunotherapy using the modified Pen a 1 peptides or molecules, for example, a vaccine expressing the modified peptides or Pen a 1 molecules.
  • modified tropomyosin used for the development of transgenic shrimp, crab, lobster, or crawfish in which the native tropomyosin production is reduced allowing for production of hypoallergenic seafood that will have reduced or abolished potential to induce allergic reactions.
  • Figure 1 shows the forty six, synthetic overlapping peptides spanning the entire sequence of Pen a 1 (length 15 amino acid residues, offset: 6 amino acids).
  • Figure 2 shows the results of the PepScan analysis of Pen a 1 peptides sera from 18 shrimp allergic subjects. (Peptide length: 15 amino acids, offset: 6 amino acids) .
  • Figure 3 shows the individually recognized epitopes and sequence comparison with allergenic and nonallergenic tropomyosins in the Pen a 1 regions 1-5.
  • Figure 4 shows the peptide amino acid sequence resulting from combinatorial substitutions and transfoiming a Pen a 1 peptide into the homologous chicken tropomyosin (TM) sequence.
  • Figure 5 is an autoradiograph showing the reactivity of the combinatorial substituted peptides with IgE from individual sera..
  • Figure 6 illustrates the amino acid positions that are critical for binding to IgE.
  • Figure 7 shows the peptides recognized by the serum IgE of six shrimp allergic subjects, each spot representing a different peptide of all 46 tested.
  • Figure 8 shows the amino acid sequence comparison of Pen a 1 with other allergenic tropomyosins.
  • Figure 9 shows the sequence comparison of IgE-binding, recombinant peptides and non-IgE-binding synthetic peptides: Identical sequences are shaded.
  • Figure 10 shows 12 Pen a 1 varieties that contain substitutions in 78 positions and will reduce or abolish the IgE antibody reactivity of the Pen a 1 molecule.
  • the invention provides highly useful compositions and methods for modifying and particularly reducing a harmful or pctentially harmful immune response against tropomyosin.
  • a harmful or pctentially harmful immune response against tropomyosin are Crustacea, arthropod, mollusk and arachnid tropomyosin, especially those of shrimp or insect origin.
  • An antigen is a molecule that elicits production of an antibody such as an IgE antibody.
  • an antibody or other similar term refers to whole immunoglobulin as well as immunologically active fragments which bind antigen.
  • the immunoglobulins and immunologically active fragments thereof include an antibody binding site.
  • Exemplary antibody fragments include for example, Fab, F(v), Fab', F(ab' fragments, "half molecules" derived by reducing the disulfide bonds of immunoglobulins, single chain immunoglobulins, or other suitable antigen binding fragments (see, e.g. Bird et al., Science, pp. 242-424 (1988); Huston et al., PNAS (USA), 85:5879 (1988); Webber et al., Mol. Immunol, 32:249 (1995)).
  • the antibody or immunologically active fragment thereof may be of animal .g. a rodent such as a mouse or rat), or chimeric form (see Morrison et al., PNAS (USA), 81 :6851 (1984); Jones et al., Nature., 321 :522 (1986)).
  • Single chain antibodies of the invention can be preferred.
  • An allergan is a subset of antigens which elicits IgE production in addition to other isotypes of antibodies.
  • An allergic reaction is one that is IgE mediated with clinical symptoms primarily involving the cutaneous (uticaria, angiodema, pruritus), respiratory (wheezing, coughing, laryngeal edema, rhinorrhea, watery/ itching eyes), gastrointestinal (vomiting, abdominal pain, diarrhea), and cardiovascular (if a systemic reaction occurs) systems.
  • An epitope is a binding site including an amino acid motif of between approximately five to fifteen amino acids which can be bound by an immunoglobulin.
  • a linear epitope is one where the amino acids are recognized in the context of a simple linear sequence.
  • a conformational epitope is one where the amino acids are recognized in the context of a particular three dimensional structure.
  • An immunodominant epitope is one which is bound by antibody in a large percentage of the sensitized population or where the titer of the antibody is high, relative to the percentage or titer of antibody reaction to other epitopes present in the same protein.
  • a decreased allergic reaction is characterized by a decrease in clinical symptoms associated with exposure to an allergen, which can involve respiratory, gastrointestinal, skin, eyes, ears and mucosal surfaces in general.
  • the first step in making the modified allergan is to identify IgE epitope binding sites and/ or immunodominant IgE binding sites.
  • the second step is to mutate one or more of the IgE binding sites, preferably including at a minimum one of the immunodominant sites.
  • the third step is to make sufficient amounts of allergan for administration to persons or animals in need of tolerance to the allergan, where the modified allergan is administered in a dosage and for a time to induce tolerance, or for diagnostic purposes.
  • the modified allergan can be administered by injection, or in some cases, by ingestion or inhalation.
  • Allergans typically have both IgE and IgG binding sites and are recognized by T cells.
  • the binding sites of the allergans can be identified using phage display libraries to identify conformational epitopes (Eichler and Houghten, (1995J Molecular Medicine Today, 1: 174-180; Jensen-Jarolim et al., (1997) J. Appl. Clin. Immunol. 101 :5153a) or by using defined peptides derived from the known amino acid sequence of the allergan (see examples below).
  • the individual peptides containing IgEbinding epitopes are mutated into the homologous sequences of the allergenic (invertebrate) to the vertebrate (non-allergenic) tropomyosins.
  • the substitutions and their effect on the IgE-binding capability of modified Pen a 1 peptides can be categorized according to different criteria. First, the minimal number of substitutions that a peptide must carry to render it non-reactive and second, the maximal number of substitutions per peptide to allow the peptide to retain at least some IgE reactivity.
  • a modified allergan will typically be made usingrecombinant techniques. Expression in a prokaryotic or eukaryotic host including bacteria, yeast, and baculovirus systems are typically used to produce large (mg) quantities of the modified allergan.
  • a modified amino acid sequence refers to an amino acid sequence of tropomyosin which has been modified by substituting an amino acid such that modified amino acid sequence binding to an IgE molecule is reduced.
  • Substituted amino acids can be selected from a nonpolar aliphatic group; a polar, uncharged group; an aromatic group; a positively charged group; or a negatively charged group.
  • crustacea as used herein, refer to shellfish such as crab, shrimp, lobster, crawfish.
  • Standard IgE antibody tests include such conventional assays such as, for example, RAST (Sampson and Albergo, 1984), ELISAs (Burks et al., 1986) immunoblotting (Burks et al. 1988), and in vivo skin tests (Sampson and Albergo, 1984). Objective clinical symptoms can be monitored before and after the administration of the modified allergan to determine any change in clinical symptoms.
  • Reference herein to a "standard IgE antibody test" or related phrase means any one of the foregoing tests, particularly the IgE ELISA test, in which at least about 45%, preferably at least about 60%, more preferably at least about 75% of the binding between the Crustacea tropomyosin and the IgE antibody has been reduced.
  • Suitable controls for performing such a test are generally known in the field and include identified IgE antibodies that bind the tropomyosin and Crustacea tropomyosin e.g., that isolated from shrimp. More particular tropomyosins and IgE antibodies for performing the test are discussed below.
  • Desensitization as used herein is defined by a sufficient decrease in IgE antibodies, as measured by the above standard IgE tests, wherein the allergic reaction of an individual manifests a decrease in clinical symptoms associated with exposure to an allergen, which can involve respiratory, gastrointestinal, skin, eyes, ears and mucosal surfaces in general.
  • a desensitizing amount as used herein is a therapeutic composition of the present invention employed in a physically discrete unit suitable as unitary dosages for a primate such as a human, each unit containing a predetermined quantity of active material calculated to produce the desired therapeuticeffect in association with the required diluent or carrier.
  • Precise desensitizing amounts of the therapeutic composition to be administered will be guided by the judgment of the practitioner, however the unit dose will generally depend on the route of administration and be in the range of 10 ng/kg body weight to 50 mg/kg body weight per day, more typically in the range of 100 ng/kg body weight to about 10 mg/kg body weight per day.
  • Transgenic animals expressing the modified allergan have two purposes.
  • the modified allergan will typically be administered in an appropriate carrier such as saline or a phosphate saline buffer.
  • the modified allergan can be administered by injection subcutaneously, intramuscularly, or intraperitoneally, by aerosol, inhaled powder, as a suppository, or by ingestion.
  • modified allergans of the invention may be administered alone, they may also be present as part of a pharmaceutical composition in mixture with conventional excipient, preferably a pharmaceutically acceptable organic or inorganic carrier substances that is generally suitable for oral or nasal delivery.
  • conventional excipient preferably a pharmaceutically acceptable organic or inorganic carrier substances that is generally suitable for oral or nasal delivery.
  • modified allergans, or combination of modified dlergans thereof can be combined with a vehicle suitable for parenteral, oral or other desired administration and which do not deleteriously react with the modified allergans and are not deleterious to the recipient thereof.
  • Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethal fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, etc.
  • the pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g.
  • lubricants preservatives, stabilizers, wetting agents, emulsifiers, slats for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously react with the modified allergans.
  • solutions preferably oily or aqueous solutions as well as suspensions, emulsions, or implants, including suppositories.
  • Ampoules are convenient unit dosages.
  • a syrup, elixir or the like can be used wherein a sweetened vehicle is employed.
  • Sustained release compositions can be formulated including those wherein the active component is protected with differentially degradable coatings, e.g., by microencapsulation, multiple coatings, etc.
  • modified allergans or combination of modified allergans, used in a giventherapy will vary according to the modified peptide or combination of peptides being utilized, the mode of application, the particular site of administration, etc. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art.
  • the nucleotide molecule encoding the modified allergan can also be administered directly to the patient, for example, in a suitable expression vector such as a plasmid, which is injected directly into the muscle or dermis.
  • the modified allergan can be expressed by a vector containing a DNA segment encoding the modified allergan.
  • Vectors can include vectors, liposomes, naked DNA, adjuvant-assisted DNA, gene gun, catheters, etc.
  • Vectors include chemical conjugates such asdescribed in WO 93/04701, which has a targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vector (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US95/02140 WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage etc.
  • the vectors can be chromosomal, nonchromosomal or synthetic.
  • Retroviral vectors include moloney murine leukemia viruses. DNA viral vectors are preferred. Viral vectors can be chosen to introduce the modified allergan to cells of choice.
  • Such vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as herpes simplex I virus (HSV) vector (Geller, A.I et al, J. Neurochem., 64:487(1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxfoid Univ. Press, Oxford, England) (1995); Geller, A.I.
  • HSV herpes simplex I virus
  • Pox viral vectors introduce the gene into the cells cytoplasm.
  • Avipox virus vectors result in only short term expression of the nucleic acid.
  • Adenovirus vectors, adeno-associated virus vectors and herpes simplex virus vectors are preferred for introducing the nucleic acid into neural cells.
  • the adenovirus vector results in a shorter term expression (about 2 months) than adenoassociated virus (about 4 months), which in turn is shorter than HSV vectors.
  • the vectors can be introduced by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include for example, naked DNA calcium phosphate precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection and viral vectors.
  • the vector can be employed to target essentially any desired target cell.
  • stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a desired location.
  • Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal, and subcutaneous injection, and oral or other known routes of administration.
  • DNA immunization employs the subcutaneous injection of a plasmid DNA (pDNA) vector encoding a specific allergenic protein.
  • the pDNA sequence is taken up by antigen presenting cells (APC).
  • APC antigen presenting cells
  • the DNA encoding allergan is transcribed and translated.
  • the allergan is then presumably presented on the surface of the APC in the context of the major histocompatibility complex (MHC) to T-cells.
  • MHC major histocompatibility complex
  • This endogenously produced allergenic protein or protein fragment induces a T phenotypic response with up-regulation of IFN-y, an increase in IgG., and suppression of allergan- specific IgE production (Speigelberg HL, Orozco EM, Roman M, et al.
  • the vector pDNA can also be conjugated to immunostimulatory sequences (ISS).
  • ISS immunostimulatory sequences
  • These ISS contain unmethylated cytosine and guanine dinucleotide repeat motifs. These CpG motifs stimulate APCs and natural killer cells to secrete IFN ⁇ and IL- 12, cytokines that promote immune deviation toward the T H I phenotype and away from the allergic TH2 phenotype (Chu RS, Targoni OS, Krieg AM, Lehman PV, Harding CV. J. Exp. Med. 1997; 186: 1623-31). These ISS stimulate immune deviation to the TH I phenotype when administered in several ways.
  • a vaccine as used herein can include any of the above viruses or vectors containing the entire nucleic acid sequence of the tropomyosin molecule, fragments thereof; modified nucleic acid sequences of the tropomyosin molecule; the entire amino acid sequence of the tropomyosin molecule or fragments thereof; modified amino acid fragments of the tropomyosin molecule or any peptides embodied in the invention.
  • the vaccine may be introduced in a suitable carrier.
  • a suitable carrier for example, sterile saline solution or sterile phosphate buffered saline.
  • the vaccine is desirably administered bysubcutaneous or intramuscular injection.
  • the treatment may consist of a single dose of vaccine or a plurality of doses over a period of time.
  • An advantageous treatment schedule requires administration of two doses of vaccine with an interval of 3 to 7, preferably 4 to 6 weeks between doses. If longer protection is required, booster doses may be administered after longer intervals, for instance after 6 months or annually.
  • booster doses may be administered after longer intervals, for instance after 6 months or annually.
  • Those who are skilled in the art may modify the vaccine regimen according to the individual patient.
  • each dose is 0.5 to 5 ml, preferably 1 to 3 ml, most preferably 2 ml of vaccine.
  • Antibodies of the invention can be prepared by techniques generally known in the art, and are typically generated to a purified crustacean tropomyosin molecule, to a modified crustacean tropomyosin molecule, preferably to peptides of a modified crustacean tropomyosin molecule or more preferably to modified peptide fragments thereof.
  • antibodies can be prepared by immunizing a mammal with the above molecules, alone or complexed with a carrier.
  • Suitable mammals include typical laboratory animals such as sheep, goats, rabbits, guinea pigs, rats and mice. Rats and mice, especially mice, are preferred for obtaining monoclonal antibodies.
  • the antigen can be administered to the mammal by any number of suitable routes such as subcutaneous, intraperitoneal, intravenous, intramuscular or intracutaneous injection.
  • the optimal immunizing interval, immunizing dose, etc. can vary within relatively wide ranges. Typical procedures involve injection of the antigen several times over a number of months.
  • Antibodies are collected from serum of the immunized animal by standard techniques and screened to find antibodies specific for tropomyosin, modified tropomyosin, modified peptides of tropomyosin and fragments thereof.
  • Monoclonal antibodies can be produced in cells which produce antibodies and those cells used to generate monoclonal antibodies by using standard fusion techniques for forming hybridoma cells. See G. Kohler, et al., Nature, 256:456 (1975). Typically this involves fusing an antibody producing cell with an immortal cell line such as a myeloma cell to produce the hybrid cell.
  • monoclonal antibodies can be produced from cells by the method of Huse et al., Science, 256: 1275 (1989).
  • One suitable protocol provides for intraperitoneal immunization of a mouse with a composition comprising the above-discussed antigens, conducted over a period of about two to seven months. Spleen cells can then be removed from the immunized mouse. Serum from the immunized mouse is assayed for titers of antibodies specific for the tropomyosin antigen selected, prior to excision of spleen cells.
  • the excised spleen cells are then fused to an appropriate homogenic or heterogenic (preferably homogenic) lymphoid cell line having a marker such as hypoxanthine-guanine phosphoribosyltransferase deficiency (HGPRT) or thymidine kinase deficiency (TK) .
  • HGPRT hypoxanthine-guanine phosphoribosyltransferase deficiency
  • TK thymidine kinase deficiency
  • Myeloma cells and spleen cells are mixed together, e.g. at a ratio of about 1 to 4 myeloma cells to spleen cells.
  • the cells can be fused by polyethylene glycol (PEG) method. See G. Kohler, et al., Nature, supra.
  • hybridoma is grown in a culture medium, e.g. RPMI-1640. See E. More, et al., J. Amer. Med. Association, 199:549 (1967).
  • Hybridomas grown after the fusion procedure are screened such as by radioimmunoassay or enzyme immunoassay for secretion of antibodies that bind to the above discussed tropomyosin antigens.
  • an ELISA is employed for the screen.
  • Hybridomas that show positive results upon such screening can be expanded and cloned by limiting dilution method.
  • the isolated antibodies can be further purified by any suitable immunological technique including affinity chromatography.
  • Tropomyosin protein including fragments and modified tropomyosins are often provide in substantially pure form. That is, the proteins have been isolated from cell substituents that naturally accompany it so that the proteins are present in at least 90 to 95% homogeneity (w/w). Proteins having at least 98 to 99% homogeneity (w/w) are most preferred for many pharmaceutical, clinical and research applications including the vaccines disclosed herein.
  • the protein should be substantially free of contaminants for therapeutic applications.
  • the proteins can be used therapeutically, or in performing in vitro or in vivo assays as disclosed herein. Substantial purity can be determined by a variety of standard techniques such as chromatography and gel electrophoresis.
  • the tropomyosins in accord with the invention can be separated and purified by appropriate combination of known techniques. These methods include, for example, methods utilizing solubility such as salt precipitation and solvent precipitation, methods utilizing the difference in molecular weight such as dialysis, ultra-filtration, gel-filtration, and SDS-polyacrylamide gel electrophoresis, methods utilizing a difference in electrical charge such as ionexchange column chromatography, methods utilizing specific affinity such as affinity chromatograph, methods utilizing a difference in hydrophobicity such as reverse-phase high performance liquid chromatograph and methods utilizing a difference in isoelectric point, such as isoelectric focusing electrophoresis, metal affinity columns such as Ni-NTA. See generally Sambrook et al.
  • specific binding or similar term is meant a molecule disclosed herein which binds another molecule, thereby forming a specific binding pair, but which does not recognize and bind to other molecules as determined by, e.g., Western blotting, ELISA, RIA, gel mobility shift assay, enzyme immunoassay, competitive assays, saturation assays or other suitable protein binding assays known in the field.
  • the modified tropomyosins of the invention can be made by one or a combination of strategies.
  • nucleotide sequences encoding tropomyosin of Crustacea or anthrop origin can be altered by mutations such as substitutions, additions or deletions that provide for functionally equivalent rucleic acid sequence having reduced antigenicity (at the protein level) .
  • Preferred mutations are substitutions in the tropomyosin regions disclosed herein.
  • a given nucleotide sequence can be mutated in vitro or in vivo, to create variations in coding regions and/ or to form new restriction endonuclease sites or destroy preexisting ones and thereby to facilitate further in vitro modification.
  • mutagenesis Any technique for mutagenesis known in the art can be used including, but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., J. Biol. Chem., 253:6551 (1978)), use of TAB Registered TM linkers (Pharmacia), PCR-directed mutagenesis, and the like.
  • the functional equivalence of such mutagenized sequences, as compared with unmutagenized sequences, can be empirically determined by comparisons of structural and/ or functional characteristics.
  • Shrimp extract from locally purchased raw brown shrimp was prepared as described previously [24].
  • Pen a 1 was purified from shrimp extract by preparative SDS-PAGE (Model 491 PrepCell, Biorad). Briefly, shrimp extract was separated on the 28 mm ID column using Laemmli discontinuous SDSPAGE buffer system [25]. A 15 mm-high stacking gel (5%T, 1.5%C) poured on top of the 65 mm-high separation gel (11%T, 1.5%C) was used to separate shrimp proteins and the fractions containing Pen a 1 were collected and pooled.
  • Pen a 1 was run by SDS-PAGE [25] and transferred onto a CNBractivated [26] nitrocellulose membrane (0.45 ⁇ m, BAS 45, Schneider and Schuell, Germany) at 0.8 mA/cm 2 for 30 min by semi-dry blotting [27]. The blots were blocked in TBS- Tween for 30 min, dried and stored between filter paper until use.
  • TBS-Tween diluted 1 : 1000 in TBSTween and washed 3 times for 10 min in TBS-Tween.
  • the membrane was washed 5 min in 37°C warm TBS-AP (0.1 M Tris-HCl, 0.1 M NaCl, 5 mM MgCb, pH 9.5) and antibody binding was visualized at using the substrate/ chromogen mixture for alkaline phosphatase at 37°C containing 450 ⁇ M 5-bromo-4-chloro-indolyl- phosphate disodium salt (BCIP; Sigma) and 400 ⁇ M nitroblue tetrazolium chloride (NBT; Sigma) solubilized in TBS-AP [28]. The reaction was stopped with deionized water and the blots were dried.
  • BCIP 5-bromo-4-chloro-indolyl- phosphate disodium salt
  • NBT nitroblue tetrazolium chloride
  • Overlapping peptides were synthesized with Fluorenylmethoxycarboyl (Fmoc) amino acids on cellulose membranes containing freehydroxyl groups according to the manufacturer's instructions of the SPOTS Epitope Mapping System (Genosys Biotechnologies, The Woodlands, TX).
  • Fmoc Fluorenylmethoxycarboyl
  • SPOTS Epitope Mapping System Genosys Biotechnologies, The Woodlands, TX.
  • NMP purified tmethyl-2 pyrrolidinone
  • Each amino acid solution was aliquoted and stored at -20°C until ready for use. Due to intrinsic instability, arginine was dissolved in NMP immediately prior to each synthesis cycle.
  • each cycle began by sterification of an Fmoc amino acid to the SPOTs cellulose membrane (Genosys Biotechnologies, Inc., The Woodlands, TX). Following incubation, the membranes were washed in N,N-didethylformamide (DMF, EM Science, Gibbstown, NJ) and 4% acetic anhydride in DMF was added to acetylate and block any uncoupled amino groups to prevent further reaction of these groups and formation of deletion sequences. After acetylation protective Fmoc groups were cleaved by incubation in 20% piperidine (Aldrich chemicals, Milwaukee, WI) in DMF, to render nascent peptides reactive.
  • DMF N,N-didethylformamide
  • the membranes were first rinsed in methanol and washed in Tris buffered saline (TBS, pH 7.5) 3 times for lOmin. The membranes were incubated in blocking solution (Genosys) diluted 1 : 10 in TBS for 2 h and then overnight with the patient's serum diluted 1 :5 with blocking buffer. After washing three times for 15 min in TBS-Tween (TBS, 0.5% Tween; pH 7.5), IgE reactivities were detected using 0.8 ⁇ Ci per membrane of 125 I-labeled horse-anti human IgE (Sanofi Diagnostics Pasteur, Chasca, MN) diluted 1 : 10 in Genosys blocking solution. Next day, the membranes were washed 3 times for 15 min in TBSTween and placed between plastic sheets and exposed to X-ray film for 72 hours.
  • TBS Tris buffered saline
  • IgE reactivities were graded according to their intensity into four categories: negative (0), weak (1), medium (2) and strong (3), and color-coded as follows: negative (white), weak intensity (light gray), medium intensity (dark gray), and strong intensity (black).
  • the intensity of the IgE reactivities was determined visually by agreement of 3 different investigators, who graded the reactivities independently assigning the above scores.
  • Table 1 shows the frequency and intensity of the IgE-recognition of the different peptides by the 18 subject's sera.
  • a wide range of peptides (from 1 to 16, mean of 8 per subject) were recognized by serum IgE of the shrimp-allergic, Pen a 1 -reactive sera.
  • none of the three control sera from shrimp allergic, Pen a 1-non reactive subjects showed IgE binding to any of the 46 peptides tested (data not shown).
  • Figure 7 shows the peptides recognized by the serum IgE of 6 shrimp-allergic subjects, with each spot representing a different peptide of all 46 tested. The number of subjects who recognized an individual peptide varied from 0 (0%) to 13 (72.2%).
  • IgE-binding peptides wsre detected over most of the tropomyosin molecule.
  • An intensity score (0-3) for each peptide was calculated by adding the individual scores obtained with the different sera (peptide score).
  • the mean intensity score of all peptides was obtained by adding all the peptide scores and dividing by 46 peptides (mean peptide score).
  • a major IgE binding region was defined as a region recognized by serum IgE from more than 9/ 18 (50%) of the subjects, and/or when the score for intensity of IgE-binding of a particular peptide was larger than mean + 1 SDEV the mean peptide score (5.9 + 6.1).
  • IgE binding regions Based on frequency and intensity of the IgE reactivities, five major IgE binding regions were identified. All five major IgE-binding regions spanned from 1 to 4 peptides, with a length from 15 to 38 amino acid residues.
  • Major IgEbinding regions identified were, region 1 : Pen a 1 (43-57), region 2: Pen a 1 (85105), region 3: Pen a 1 (133-148), region 4: Pen a 1 (187-202) and region 5: Pen a 1 (247284).
  • Region 1 is recognized by 10/ 18 (55.5%) subjects, region 2 by 15/ 18 (83.3%), region 3 by 10/ 18 (55.5%), region 4 by 5/ 18 (27.5%) and region 5 by 12/ 18 (66.6%).
  • the score for intensity of IgE recognition was 18, 19, 20, 12, and 12.2 for the five regions, respectively.
  • IgE-binding epitopes were identified using 5 to 9 amino acids-long peptides with an offset of two amino acids. If no or only minimal reactivity was detected the peptide size was increased to 9 to 15 amino acid residues.
  • the peptides that were found to be IgE-binding within regions 1 to 5 are also summarized in table 2.
  • Figures 3.1 to 3.5 show the identified strongest individual IgE-binding reactivities (epitopes) of regions 1 to 5, common epitope cores, and sequence comparisons with allergenic and nonallergenic tropomyosins (Crustacea: Penaeus aztecus (brown shrimp, Pen a 1) , Metapenaeus ensis (greasy-back shrimp, Met e 1), Homarus ame ⁇ canus (Atlantic lobster, Horn a 1), H. americanus slow muscle tropomyosin (HomaTMs), Panulirus stimpsoni (spiny lobster, Pan s 1).
  • allergenic and nonallergenic tropomyosins Crustacea: Penaeus aztecus (brown shrimp, Pen a 1) , Metapenaeus ensis (greasy-back shrimp, Met e 1), Homarus ame ⁇ canus (Atlantic lobster, Horn a 1), H. americanus slow muscle tropomyo
  • Insecta Pe ⁇ planeta americana (American cockroach, Per a 7), Arachnida: Dermatophagoides pteronyssinus, D. fa ⁇ nae (house dust mites, Der p 10, Der f 10).
  • Vertebrata Gallus gallus (chicken, alpha-tropomyosin Galg ⁇ TM; beta-tropomyosin Galg ⁇ TM), Sus scrofa (pig, Suss ⁇ TM), Salmo trutta (Atlantic salmon, SaltTM), Oryctolagus cuniculus (rabbit, Oryc ⁇ TM) (GenBank data)).
  • Pen a 1 87-101 ANRRIQLLEEDLER
  • Pen a 1 91-101 RIQLLEEDLER
  • This core sequence is identical with those of other Crustacea and Per a 7, whereas Der p 10 carries one substitution in position 95. All vertebrate tropomyosins carry the same three substitutions in positions 95, 98, and 100.
  • Region 3 Six out of seven subjects tested reacted with peptides in region 3 (Pen a 1
  • the size of the individual epitope ranged from 6 to 9 amino acid residues.
  • the core epitope of region 3 was Pen a 1 137- 141 (DEERM) which is recognized by all 6 subjects and is identical to the homologous sequences of American cockroach (Periplaneta ame ⁇ can ⁇ ] and house dust mite (Dermatophagoides pteronyssinus) allergenic tropomyosins, Per a 1 and Der p 10, respectively.
  • the comparison with vertebrate tropomyosin shows a substitution in position 141 where an arginine (R) is substituted for a lysine (K) residue.
  • Region 4 All individual epitopes of the three subjects tested varied in length from 11 to
  • region 5 (Pen a 1 247-284) three epitopes were identified. All four subjects reacted with Pen a 1 266-273 (KYKSITDE). This sequence was for all four subjects the minimal IgE-binding site; no other peptide showed stronger reactivity.
  • This epitope differs from both allergenic, arthropod and nonallergenic, vertebrate tropomyosins.
  • the second epitope of region 5, is centered around a core, Pen a 1 251-259 and is recognized by 3/4 subjects; the core does not differ for the homologous sequences of Per a 1 or Der p 10 but differs in three positions from non-allergenic, vertebrate tropomyosins.
  • H. americanus slow muscle tropomyosin (HomaTMs) Panulirus stimpsoni (spiny lobster, Pan s 1)
  • Insecta Periplaneta americana (American cockroach, Per a 7)
  • Arachnida Dermatophagoides pteronyssinus, (house dust mite, Der p 10).
  • Vertebrata Gallus gallus (chicken alpha-tropomyosin, Galg ⁇ TM)
  • G. gallus (chicken beta- tropomyosin, chicken Galg ⁇ TM), Sus scrofa (swine alpha tropomyosin, Suss TM), Salmo trutta (Atlantic salmon, SaltTMl, SaltTM2) Oryctolagus cuniculus (rabbit alpha-tropomyosin, Oryc ⁇ TM)
  • the combinatorial substitution analysis was performed with individual sera rather than serum pool.
  • the substitutions and their effect on the IgE-binding capability of modified Pen a 1 peptides can be categorized according to different criteria. First, the minimal number of substitutions that a peptide must carry to render it nonreactive and second, the maximal number of substitutions per peptide allow the peptide to retain at least some IgE reactivity. In all 17 epitope-subject combinations that were studied by mutational analysis, one substitution may be sufficient to render Pen a 1 epitopes non-IgE binding.
  • Table 5 lists all amino acid substitutions in Pen a 1 epitopes 1, 2, 3a, 3b, 5a, 5b, and 5c that result in complete loss or reduction of IgE antibody reactivity.
  • the first column lists all the sequences that carry substitutions that abolished IgE antibody reactivity with any of the sera that were used to analyze that particular epitope.
  • the second column lists all the sequences that carry substitutions which in some cases reduce or abolish IgE antibody reactivity with the sera that were used to analyze the previously unaltered Pen a 1 epitopes. The sequences are named according to the substitutions they carry (King et al., Allergan
  • Pen a 1 44 is a Pen a 1 sequence that has an I (Isole cine) in Pen a 1 position 44. This Isoleucine replaces a H (Histidine) that can be found in position 44 in the unaltered Pen a 1 sequence (see row labeled as "Pen a 1 sequence") .
  • the first critical position is located at position 142 and an Aspartate (D) is replaced with Glutamate (E); it abolished the IgE antibody reactivity of four of the five epitope 3a- reactive sera.
  • the second critical position is position 136; a Serine (S) has to be replaced with a Lysine (K) to abolish the IgE antibody reactivity of three subjects' sera.
  • the critical position of epitope 3b is position 144; a mutation from a Leucine (L) to a Glutamine (Q) abolished the IgE antibody reactivity of both subjects' sera tested.
  • the relative frequency of different amino acids in the tropomyosin molecule relative to the main IgE-binding regions were analyzed.
  • Five categories of amino acids were considered: non-polar, aliphatic (alanine A, glycine G, isoleucine I, leucine L, proline P, valine V; polar, uncharged (cystehe C, asparagine N, methionine M, glutamine Q, serine S, threonine T; aromatic (phenylalanine F, tryptophane W, tyrosine Y); positively charged (histidine H, lysine K, arginine R) and negatively charged (aspartic acid D, glutamic acid E).
  • the frequency of the different groups of amino acids in each IgE-binding region was considered in relation to the number of amino acids present in that group, supposedly that all amino acids have the same probability to appear in a protein (probability l).For the whole molecule, negatively charged amino acids are 2.5 times more frequent than would be expected by chance. Contrarily, aromatic residues are almost absent in the tropomyosin molecule. The other three groups of amino acids are present in the molecule with the frequency expected considering all 20 amino acids equally probable. In the five main IgE-binding regions the frequency of each amino acid group is the same as the frequency observed in the whole molecule. No substantial differences in amino acid group composition in the five IgE-binding regions compared to the whole molecule were detected.
  • Region 1 is identical within crustaceans but differs from other invertebrates and vertebrates, with only 26%,60% or 33% identity with Schistosoma, insects and vertebrates respectively.
  • regions 2 and 4 show 100% similarity with homologous regions of arthropod tropomyosins from American cockroach Periplaneta americana (Per a 7), fruit fly Drosophila
  • regions 2 and 4 show significant similarity with corresponding sequences in other tropomyosins, including those from vertebrates (up to 85 %).
  • Regions 3 and 5 are identical within crustaceans, and the identity with tropomyosins of other arthropods reaches up to 89%.
  • Pen a 1 regions differ substantially from those of homologous sequences (as low as 40% identity) in helminths and vertebrate tropomyosins.
  • FIG. 8 shows the amino acid sequence comparison of Pen a 1 with other allergenic tropomyosins, whose B cell epitopes have been partially characterized such as Pen i 1 from the shrimpPen ⁇ eus indicus, Tur c 1 from the snail Turbo cornutus, and Cra g 1 from the oyster Crassotrea gigas.
  • peptide fragments containing T-cell epitopes of bee venom phospholipase A2 intact, whose B-cell epitopes have been modified to abolish IgE binding, have been successfully used for immunotherapy with a lower risk of reactions [34]. Therefore, identification and subsequent modification of the Bcell epitopes of tropomyosin could serve as the basis for the development of new safer immunotherapy for food allergy, and as well as for the introduction of new hypoallergenic foods.
  • Pen a 1 IgE-binding regions have been identified using synthetic overlapping peptides, 15 amino acids long with offset of 6, spanning the whole length of Pen a 1.
  • the five IgE-binding regions are distributed along the molecule at approximately every 42 amino acid residues. This results suggest a relation with the heptameric repeat pattern characteristic for the - helical, coiled-coil structure of tropomyosin [21].
  • the five regions identified contain at least 15 amino acid residues (region 5 spans 37 residues).
  • Regions 2 and 5 seem to be of particular importance, since the homologous sequences in oyster (Cra g 1) and in the snail (Tur c 1) tropomyosins bind IgE antibodies of mollusk-allergic subjects, thus supporting the notion that tropomyosin is the cause of clinically relevant cross- sensitization between crustaceans and mollusks [24, 35].
  • Pen a 1 epitopes In order to characterize Pen a 1 epitopes, a recombinant peptide library (Novatope epitope mapping system, Novagen) was constructed. The Pen a ⁇ coding plasmid was randomly cleaved by DNase I in the presence of Mn 2+ causing double strand cleavage. Electrophoretically separated fragments, averaging 50 to 150 bp in size, were eluted (QIAEX II Agarose Gel Extraction Kit, Qiagen), treated successively with T4 DNA polymerase and Tth DNA polymerase, ligated into the pTOPE T vector, and transfected into NovaBlue (DE3) cells. The library was screened with a sera pool of shrimp- allergic subjects and positive clones sequenced.
  • the coupling reaction is monitored visually by staining the free amines after each coupling cycle with bromophenol blue.
  • the resulting peptides are covalently bound to the membrane at their C- terminus.
  • Each synthesis cycle begins with esterifying the appropriate Fmoc amino acid to the cellulose membrane or the previous amino acid.
  • the coupling reactions are followed by acetylation with acetic anhydride in N,Ndimethylformamide to render the peptides unreactive during subsequent cycles.
  • the Fmoc protective groups are removed by adding piperidine to activate the nascent peptides. To add the remaining amino acids the same cycle of coupling, blocking, and deprotection is repeated until the desired peptides are generated.
  • the side chains are then deprotected with a 20:20: 1 mixture of dichloromethane, trifluoroacetic acid, and triisobutylsilane and washed with methanol.
  • the membranes are stored at-20°C until used.
  • the synthesis schedules can be calculated using the software provided by Genosys or using the graphing calculator HP48GX.
  • the advantage of the HP48GX printouts is that the positions of a particular amino acid are provided as graphs rather than lists of position numbers.
  • the membranes were blocked and incubated with 1 :5 diluted serum pool or individual sera of shrimp- allergic subjects overnight.
  • IgE reactivities were detected, either using 10 ml 125 I-labeled horse-anti-IgE (0.08 ⁇ Ci/ml; Sanofi Diagnostics Pasteur, Inc.) or monoclonal alkaline phosphatase- labeled anti-human-IgE (Southern Biotechnology Associates, Birmingham, AL, USA) and autoradiography.
  • the exposure time for 125 I-labeled anti-IgE was 72 h.
  • blots were washed with freshly prepared assay buffer (100 mM diethanolamine/HCI, 1.0 mM MgCt pH 10.0), incubated in 1:50 diluted Nitroblock ® chemiluminescence enhancer (Tropix, Bedford, MA) for 5 min and incubated in a 1 : 1000 dilution of CSPD (disodium 3-(4-methoxy-spiro ⁇ dioxetane-3, 2'-(5'chloro) tricyclo[3.3.1.1. 3 . 7 ]decan ⁇ -4-yl)phenyl phosphate; Tropix) for 5 min. Excessive liquid was drained, and the blots were sealed between transparencies and exposed to autoradiography film for 15, 30, 60 and 120 sec.
  • assay buffer 100 mM diethanolamine/HCI, 1.0 mM MgCt pH 10.0
  • Nitroblock ® chemiluminescence enhancer Tropix, Bedford, MA
  • CSPD
  • Table 7 shows all the substitutions (position, pos; substituting amino acid, aa) that can be considered to reduce or abolish the IgE reactivity of major and minor IgE-binding regions.
  • minor IgE reactive regions defined as regions to which at least one allergic subject shows strong IgE antibody reactivity.
  • the Example shows that the methods may be used to identify IgE-binding sequences of food allergans, and the SPOTs procedure l ⁇ sulted in the identification of more epitopes of the major shrimp allergan Pen a 1.
  • the sequences of the synthetic, overlapping peptides have a defined offset, epitopes that are located on two peptides overlapping may not always be readilyfound.
  • the usage of 125 I-labeled detection antibody seems to be superior over enzyme-labeled anti IgE antibodies.
  • the regeneration of SPOTs membranes is possible, but it is prudent to test regenerated membranes for residual activity. We synthesize new peptides for each experiment.
  • an advantage of the recombinant library method is that the peptide length is not limited to 15 residues as it is the case for the SPOTs system which may allow the identification of at least some conformational epitopes.
  • An additional advantage of the Novatope system that it is easy to test additional patients' sera by simply growing more peptide-expressing E.coli and use lysates in a dot blot or grid blot. Synthetic peptides have to be resynthesized which requires in comparison a much higher experimental effort.
  • a major advantage of synthetic peptides is the ease in which the impact of amino acid substitutions have on the IgE binding of epitopes [38,39,40]; the side-by-side comparison of unmodified and mutated epitopes allows an easy quantification of changes of protein structure on the alleiganicity of proteins [39]. This approach may be used to produce foods and other allergans with reduced allerganicity.
  • the synthesis protocol uses an acetylation step at the end of each cycle to acetylate any unreacted free amines with acetic anhydride. This prevents them from coupling to any subsequent amino acids and virtually eliminates the synth ⁇ is of deletion sequences.
  • the purity of the peptides synthesized varies for each peptide and is dependent upon sequence and length even though the peptide purity is typically larger than 70% (Genosys, personal communication). As a consequence it is essential to verify the results obtained with overlapping peptides with highly purified peptides when peptides are designed for critical applications such as allergan-specific immunotherapy.
  • DNA sequences coding for modified and unmodified Pen a 1, Pen a 1 fragments, or Pen a 1 peptides will be administered subcutaneous , intramuscular, or orally. Dosage, frequency and duration of treatment will be adjusted on a individual basis.
  • DNA immunization will employ naked DNA, plasmid DNA (pDNA) vectors encoding for modified and unmodified Pen a 1, Pen a 1 fragments, or Pen a 1 peptides as well as pDNA conjugated to immunostimulatory sequences (ISS). These ISS contain unmethylated cytosine and guanine dinucleotide repeat motifs.
  • DNA molecules, plasmid DNA and ISS will be administered in several ways. They can be administered with DNA encoding the allergen (DNA immunization), given alone, or conjugated with modified and unmodified allergen, allergen fragments, and peptides.
  • Modified and unmodified Pen a 1 , Pen a 1 fragments, or Pen a 1 peptides will be used to produce specific antibodies in mice, rats, rabbits or other experimental animals.
  • the produced sera and monoclonal antibodies will be used to detect, measure and standardize modified and unmodified Pen a 1 , Pen a 1 fragments, or Pen a 1 peptides, that are produced for diagnostic and therapeutic purposes.
  • monoclonal antibodies are especially useful as secondary standards, since monoclonal antibodies recognize only specific determinants (epitopes) of allergens and non-allergens; the specificity of an antibody depends on the uniqueness of the epitope.
  • these antibodies and antisera will also be very useful to characterize cross-reacting epitopes on related and unrelated proteins.
  • Example 1 Transgenic Crustacea
  • the DNA coding for the mutated tropomyosins will be developed by site- directed mutagenesis of Pen a 1.
  • SMI spermatophore-microinjection
  • the mutated Pen a 1 will introduced into the genome of shrimp or prawn species such as the giant freshwater prawn
  • Macrobrachium rosenbergii or Tiger shrimp (Penaeus monodon) .
  • Approximately 1 ⁇ g of the circular plasmid DNA will be directly microinjected into spermatophores. Fertilization and hatching of shrimp or prawns created with SMI were completed in vivo. The genomes of free swimming, SMI-created larvae (21 days after fertilization) will be analyzed by PCR and Southern blot analyses.
  • any of the modified tropomyosins disclosed herein including particular modified shrimp tropomyosins can be transduced into shrimp to make the transgenic shrimp.
  • Such shrimp may or may not include tropomyosin "knock-out" mutations as needed for a particular application.
  • Such transgenic shrimp will be especially useful in seafood farming applications featuring shrimp with reduced or negligible allergen potential.

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Abstract

L'invention concerne des compositions et des procédés permettant de modifier une réponse immunitaire contre la tropomyosine et des antigènes associés. Un mode de réalisation concerne des vaccins comprenant de la tropomyosine modifiée réduisant ou éliminant une réponse immunitaire non souhaitée contre la tropomyosine. L'invention concerne également des molécules de tropomyosine modifiée présentant des sites de liaison (épitope) d'anticorps modifiés qui réduisent ou éliminent de manière significative la propension à déclencher le système immunitaire. L'invention concerne également des animaux transgéniques, en particulier des crevettes, porteurs d'au moins l'une des molécules de tropomyosine modifiée susmentionnées. Les procédés décrits dans la présente invention peuvent servir à de multiples applications, y compris, à la réduction des réponses immunitaires nuisibles chez les crustacés, les anthropodes et chez d'autres animaux.
PCT/US2000/030968 1999-11-12 2000-11-12 Compositions et procedes permettant de modifier une reponse immunitaire contre la tropomyosine WO2001034186A1 (fr)

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EP1269837A2 (fr) * 2001-06-08 2003-01-02 TaiMont Biotech, Inc. Lait contenant un allergène pour le traitment des allergies
US7060687B2 (en) 2001-02-07 2006-06-13 Genmont Biotechnology Co. Live vaccines for allergy treatment
US7151163B2 (en) * 2003-04-28 2006-12-19 Sequoia Pharmaceuticals, Inc. Antiviral agents for the treatment, control and prevention of infections by coronaviruses
EP2144063A1 (fr) * 2007-04-06 2010-01-13 Maruha Nichiro Seafoods, Inc. Procédé de détection hautement sensible de protéines alimentaires
US8021868B2 (en) 2007-01-05 2011-09-20 Promd Biotech Co., Ltd. Anti-allergy lactic acid bacteria
US20170296652A1 (en) * 2014-09-17 2017-10-19 Stc.Unm Methods and compositions involving recombinant hypoallergens
US9920100B2 (en) 2015-06-05 2018-03-20 The Chinese University Of Hong Kong Mimotopes of tropomyosin for use in immunotherapy for shellfish and/or arthropod allergy
CN111596070A (zh) * 2020-06-10 2020-08-28 宁波大学 一种三疣梭子蟹原肌球蛋白过敏检测试剂的应用
CN112142836A (zh) * 2019-06-28 2020-12-29 中国食品发酵工业研究院有限公司 一种原肌球蛋白抗原的表位肽及其应用

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7060687B2 (en) 2001-02-07 2006-06-13 Genmont Biotechnology Co. Live vaccines for allergy treatment
EP1269837A3 (fr) * 2001-06-08 2003-09-24 TaiMont Biotech, Inc. Lait contenant un allergène pour le traitment des allergies
EP1269837A2 (fr) * 2001-06-08 2003-01-02 TaiMont Biotech, Inc. Lait contenant un allergène pour le traitment des allergies
US7151163B2 (en) * 2003-04-28 2006-12-19 Sequoia Pharmaceuticals, Inc. Antiviral agents for the treatment, control and prevention of infections by coronaviruses
WO2005032453A3 (fr) * 2003-04-28 2007-02-08 Sequoia Pharmaceuticals Inc Agents antiviraux destines au traitement, a la regulation et a la prevention d'infections a coronavirus
JP2007526213A (ja) * 2003-04-28 2007-09-13 セコイア、ファーマシューティカルズ、インコーポレイテッド コロナウイルスによる感染症の治療、管理および予防用の抗ウイルス剤
US8021868B2 (en) 2007-01-05 2011-09-20 Promd Biotech Co., Ltd. Anti-allergy lactic acid bacteria
EP2144063A1 (fr) * 2007-04-06 2010-01-13 Maruha Nichiro Seafoods, Inc. Procédé de détection hautement sensible de protéines alimentaires
EP2144063A4 (fr) * 2007-04-06 2010-04-28 Maruha Nichiro Seafoods Inc Procédé de détection hautement sensible de protéines alimentaires
US8377696B2 (en) 2007-04-06 2013-02-19 Maruha Nichiro Seafoods, Inc. Highly sensitive method for detecting protein in food
US20170296652A1 (en) * 2014-09-17 2017-10-19 Stc.Unm Methods and compositions involving recombinant hypoallergens
US9920100B2 (en) 2015-06-05 2018-03-20 The Chinese University Of Hong Kong Mimotopes of tropomyosin for use in immunotherapy for shellfish and/or arthropod allergy
CN112142836A (zh) * 2019-06-28 2020-12-29 中国食品发酵工业研究院有限公司 一种原肌球蛋白抗原的表位肽及其应用
CN111596070A (zh) * 2020-06-10 2020-08-28 宁波大学 一种三疣梭子蟹原肌球蛋白过敏检测试剂的应用

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