IMMUNOGENIC PRODUCT COMPRISING AN IgE FRAGMENT FOR TREATING IgE-MEDIATED INFLAMMATORY DISORDERS

Abstract

An immunogenic product including at least one immunoglobulin or fragment thereof conjugated with a carrier protein, wherein the at least one immunoglobulin is IgE and preferably wherein the IgE fragment includes the IgE Cε3 domain, and wherein the carrier protein is preferably CRM.sub.197. Also the use of this immunogenic product for treating inflammatory disorders, and in particular allergic disorders.

Claims

1-16. (canceled)

17. An immunogenic product comprising at least one immunoglobulin or immunoglobulin fragment conjugated with a carrier protein, wherein the at least one immunoglobulin is IgE, and wherein the IgE fragment comprises the IgE Cε3 domain.

18. The immunogenic product according to claim 17, wherein the carrier protein is CRM.sub.197.

19. The immunogenic product according to claim 17, wherein the immunoglobulin fragment comprises a part or the totality of the IgE Cε3 and Cε4 domains.

20. The immunogenic product according to claim 17, wherein the immunoglobulin fragment comprises a part or the totality of the IgE Cε2, Cε3 and Cε4 domains.

21. The immunogenic product according to claim 17, wherein the IgE or the fragment thereof comprises the G335C mutation.

22. The immunogenic product according to claim 17, wherein the IgE fragment comprises or consists in SEQ ID NO:7.

23. The immunogenic product according to claim 17, wherein the IgE or the IgE fragment comprises at least one glycosylation.

24. A composition comprising the immunogenic product according to claim 17.

25. The composition according to claim 24, being a pharmaceutical composition and comprising at least one pharmaceutically acceptable excipient.

26. The composition according to claim 24, being a vaccine composition and comprising at least one adjuvant.

27. The composition according to claim 24, being an emulsion.

28. A method for producing an immunogenic product according to claim 17, the method comprising steps of: (a) contacting the immunoglobulin or fragment thereof with a heterobifunctional crosslinker containing a NHS-ester, thereby obtaining a complex between a heterobifunctional crosslinker containing a NHS-ester and the immunoglobulin or fragment thereof; (b) contacting the carrier protein with a heterobifunctional crosslinker containing a NHS-ester to generate a complex between the heterobifunctional crosslinker containing a NHS-ester and the carrier; and (c) contacting the complex between a heterobifunctional crosslinker containing a NHS-ester and the immunoglobulin or fragment thereof obtained at step (a) with the complex between the heterobifunctional crosslinker containing a NHS-ester and the carrier obtained at step (b).

29. The method according to claim 28, the method comprising steps of: (a) contacting the immunoglobulin or fragment thereof with N-[γ-maleimidobutyryloxy]-succinimide ester (sGMBS), thereby obtaining a sGMBS-immunoglobulin or fragment thereof complex; (b) contacting the carrier protein with N-succinimidyl-S-acetylthioacetate (SATA) to generate a carrier-SATA complex; and (c) contacting the sGMBS-immunoglobulin or fragment thereof complex obtained at step (a) with the carrier-SATA complex obtained at step (b).

30. A method for treating an inflammatory disorder in a subject, comprising administering to the subject an immunogenic product according to claim 17.

31. The method according to claim 30, wherein the inflammatory disorder is associated with aberrant IgE expression or activity.

32. The method according to claim 30, wherein the inflammatory disorder is selected from the group comprising asthma, allergic conditions, anaphylaxis, atopic disorders, bullous pemphigoid, respiratory disorders, nasal polyposis and other conditions involving airway inflammation; inflammatory and/or autoimmune disorders or conditions, gastrointestinal disorders or conditions; systemic lupus erythematosus; mastocytosis and mast cell activation syndrome (MCAS).

33. The method according to claim 30, wherein the inflammatory disorder is selected from the group comprising food allergies, venom allergy, allergy to animals, drug allergy, hyper IgE syndrome, allergic rhinitis, allergic conjunctivitis and allergic enterogastritis, urticaria, eczema, asthma, allergic bronchopulmonary aspergillosis, allergic bronchopulmonary mycosis, eosinophilia, fibrosis and excess mucus production systemic sclerosis inflammatory bowel diseases (IBD), eosinophilic esophagitis (EE), eosinophilic-mediated gastrointestinal disease, ulcerative colitis and Crohn's disease.

34. The method according to claim 30, wherein the inflammatory disorder is selected from allergy, anaphylaxis, allergic asthma, allergic rhinitis, allergic conjunctivitis, nasal polyposis.

35. The method according to claim 30, wherein the inflammatory disorder is food or venom allergy.

36. The method according to claim 30, wherein the method is for inducing desensitization of a subject allergic to a specific antigen, wherein said immunogenic product or composition and said specific antigen are administered to the allergic subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0267] FIG. 1 shows the generation of hIgE Kinoid (hIgE-K). (A) Synthesis of hIgE-K using a thiol-maleimide conjugation. (B) Generation of high molecular weight kinoids upon conjugation of the IgE Cε3-Cε4 fragment to CRM.sub.197 was confirmed using SDS-PAGE and (C) HPLC.

[0268] FIG. 2 shows the neutralization of anti-hIgE antibodies by hIgE-Kinoid. (A) Intra-muscular vaccination protocol outline. hIgE.sup.KI mice (which express human IgE instead of mouse IgE) were vaccinated with hIgE-K (or CRM.sub.197 alone as control), emulsified with the adjuvant Squalene-in-water emulsion (SWE). (B) Anti-hIgE and (C) anti-CRM.sub.197 antibody titers in sera at 5, 9, 21, 30 and 39 weeks after first injection of kinoid. Results show values from individual mice with bars indicating medians. (D) Anti-hIgE neutralizing capacity in sera collected at week 5. Bone marrow-derived cultured mast cells (BMCMCs) expressing the hIgE receptor FcεRI were derived from mice humanized for FcεRI. BMCMCs were pre-incubated with sera from mice vaccinated with hIgE-K (collected 39 days after the first injection of vaccine) at the indicated dilution. Immediately after, fluorescently-labeled (FITC) hIgE were added for 30 min Cells were washed and levels of FITC fluorescence on BMCMCs was quantified by flow cytometry. (E) Levels of total hIgE in sera collected at week 5, 9, 21, 30 and 39. Results show values from individual mice with bars indicating mean±SEM. (B-E) Data are from a single experiment with n=8 mice per group, representative of two independent experiments. ***, P<0.001 (Mann-Whitney U test).

[0269] FIG. 3 shows that vaccination with hIgE-K prevents IgE-mediated systemic anaphylaxis. (A) Protocol outline. IgE/FcεRI humanized mice (which express human IgE and human IgE receptor FcεRI) were vaccinated (intra-muscular, i.m) with hIgE-K (or CRM.sub.197 alone as control), emulsified with the adjuvant SWE. At week 9, mice were sensitized with hIgE anti-nitrophenyl (NP) and challenged one day later with NP (nitrophenyl) coupled to BSA both in intra-venous, as indicated. (B) Antibody titers in sera 5 weeks after first injection of kinoid. Results show values from individual mice with bars indicating medians±SEM. (C) Changes in body temperature at week 0, 1 and 3 (Δ° T, mean±SEM) after mice injection with IgE-K or CRM.sub.197. Data are pooled from two independent experiments with a total of n=7-9 mice per group. (D) Changes in body temperature (which is used as a main readout of anaphylaxis in mice) (Δ° T, mean±SEM) after intravenous injection of 10 μg anti-NP hIgE. Data are pooled from two independent experiments with a total of n=7-9 mice per group. (E) Changes in body temperature (Δ° T, mean±SEM) after intravenous injection of 500 μg of NP-BSA. Data are pooled from two independent experiments with a total of n=7-9 mice per group. *, ** or ***, P<0.05, 0.01, or 0.001 (Mann-Whitney U test).

[0270] FIG. 4 shows that in a genetically predisposed allergic mouse model, vaccination with hIgE-K prevents IgE-mediated systemic anaphylaxis. (A) Protocol outline. IgE/FcεRI humanized mice bearing a F709 IL4Ra mutation (the equivalent mutation has been linked to atopy in human, and the mutation is known to increase susceptibility to IgE-mediated anaphylaxis in mice) were vaccinated with hIgE-K (or CRM.sub.197 alone as control), emulsified with the adjuvant SWE. At week 6, mice were injected i.v. with 250 μg of anti-hIgE. (B) Changes in body temperature (which is used as a main readout of anaphylaxis) (Δ° T, mean±SEM) after intravenous injection of 250 μg of anti-hIgE Abs. (C) Survival curve after intravenous injection of 250 μg anti-hIgE. Data are pooled from two independent experiments with a total of n=9 mice per group. ** or ***, P<0.01, or 0.001 (Mann-Whitney U test).

EXAMPLES

[0271] The present invention is further illustrated by the following examples.

[0272] The present invention relates to an immunogenic product using CRM.sub.197 as a carrier protein. The properties of the immunogenic product of the invention are illustrated by the following examples.

[0273] CRM.sub.197 is a non-toxic form of diphtheria toxin without toxic activity due to a single base substitution, in its toxin domain, from glycine to glutamate in position 52 (Uchida et al., 1973 J Biol Chem).

[0274] A thiol-maleimide conjugation is employed for the preparation of IgE based immunogenic products. Sulfhydryl moieties were introduced on the carrier protein CRM.sub.197 with SATA and subsequent hydroxylamine deprotection, while IgE or the fragment of IgE was derivatized by sGMBS, a maleimide-containing agent. Both SATA and sGMBS are heterobifunctional crosslinkers containing a NHS-ester, which reacts with primary amines (such as ε-amino groups of lysine residues and protein N-termini).

Example 1: Anti-IgE Vaccination Prevents Human IgE-Mediated Severe Allergic Reactions in Humanized Mice

Materials and Methods

Mice

[0275] hIgE.sup.KI mice were obtained inserting human IgE sequence (1080 base pair, located on human chromosome 14: 106,064,224-106,068,065) on mouse chromosome 12 (Chr12:113,147,778). IgE/FcεRI humanized mice were generated by intercrossing of hIgE.sup.KI and mFcεRI.sup.−/− hFcεRI.sup.Tg mice (Dombrowicz D et al., Anaphylaxis mediated through a humanized high affinity IgE receptor. Journal of immunology (Baltimore, Md: 1950). 1996; 157(4):1645-51). IgE/FcεRI humanized mice bearing the F709 IL4Ra mutation were generated by intercrossing of IgE/FcεRI humanized mice with F709 IL4Ra mice (Tachdjian R et al., In vivo regulation of the allergic response by the IL-4 receptor alpha chain immunoreceptor tyrosine-based inhibitory motif. J Allergy Clin Immunol. 2010; 125(5):1128-36.e8). Mice were maintained in a specific pathogen-free facility at Institut Pasteur. Mice were bred at Institut Pasteur and demonstrated normal development and breeding patterns. All animal care and experimentation were conducted in compliance with the guidelines and specific approval of the Animal Ethics committee CETEA (Institut Pasteur, Paris, France) registered under #170043, and by the French Ministry of Research.

IgE Fragments Production

[0276] The recombinant hIgE Cε3-4 fragment (containing G335C mutation, with C-terminal Strep Twin tag and harboring the amino acid sequence of SEQ ID NO: 7) was synthesized and transiently transfected into exponentially growing Expi-293 cells that were cultured in Expi293™ Expression Medium (Life Technologies) in suspension at 37° C. in a humidified 5% CO2 incubator on a shaker platform rotating at 110 rpm. Twenty-four hours before transfection, cells were harvested resuspended in Expi293™ Expression Medium at a density of 2×10.sup.6 cells/ml, and cultured overnight in the same conditions as mentioned above. Twenty-four hours after, 500 μg of expressing plasmids and 1350 μL of Expifectamine were pre-incubated during 5 min in Opti-MEM (Life Technologies) medium and mixed together. After 20 minutes of incubation, the mixture is added to Expi-293 cells at density of 2.9×10.sup.6 cells/mL. Twenty hours after the transfection, 25 mL and 2.5 mL of transfection enhancer 1 and 2 (ThermoFisher) respectively were added. Cells were cultured for 5 days after transfection, supernatants were harvested, centrifuged at 4200 rpm for 30 min and filtered (0.2 μm). Proteins were purified by affinity chromatography using an AKTA pure FPLC instrument (GE Healthcare) and Strep-Tactin® Column (IBA Lifescience).

Synthesis and Characterization of hIgE Kinoid

[0277] hIgE Cε3-4 was modified with N-γ-maleimidobutyryl-oxysuccinimide ester (sGMBS; Thermo Fisher), a maleimide-containing agent reacting with primary amines. Buffer of hIgE Cε3-4 was exchanged against modification buffer (70 mM Phosphate buffer, 150 mM NaCl, 5 mM EDTA, pH=7.2) at 1 mg/mL. A solution of 10 mM of sGMBS was prepared and added to the hIgE Cε3-4 at a 1:30 ratio and incubated during 60 minutes at room temperature (protected from light). Excess sGMBS was removed and buffer exchanged against modification buffer using Zeba desalting spin column (Thermo Fisher). CRM.sub.197 was purchased from Pfenex (USA). Sulfhydryl moieties were introduced on the carrier protein CRM.sub.197 with SATA (N-succinimidyl-S-acetylthioacetate.). CRM.sub.197 was diluted in modification buffer at 2 mg/mL and a freshly prepared solution of 100 mM SATA (dissolved in DMSO) was added at a 1:80 molar ratio and incubated 30 minutes at room temperature (protected from light). Excess SATA was removed and buffer exchanged against modification buffer using Zeba desalting spin column SATA modified CRM.sub.197 was incubated with a solution of hydroxylamine at a 50 mM final concentration, at room temperature for 120 minutes, protected from light. Excess hydroxylamine was removed and buffer exchanged against modification buffer using Zeba desalting spin column After CRM.sub.197 and hIgE Cε3-4 functionalization, protein content of each preparation was determined by Bradford (Thermo Fisher) assay according to manufacturer's instructions.

[0278] Functionalized CRM.sub.197 was added to functionalized hIgE Cε3-4 at a molar ratio of 1:1 and a final concentration of 0.4 mg/mL. The mixture was incubated 16 hours at 4° C., protected from light, and subsequently buffer exchanged against modification buffer using Zeba desalting spin column. Protein content was determined by Bradford assay. Resulting hIgE kinoid (hIgE-K) was then 0.22 μm sterile filtered and stored at 4° C.

[0279] The hIgE-K was characterized using different in vitro methods. To analyze the profiles of the kinoids obtained, SDS-PAGE and western blot were performed against the hIgE Cε3-4 fragment (Strep-TACTIN HRP conjugate (IBA Lifescience)). Size exclusion (SE)-HPLC using a Bio SEC-5 column (2000 Å, 5 μm, 7.8*300 mm, Agilent) and Bio SEC-3 column (300 Å, 3 μm, 7.8*300 mm, Agilent) was also used. SE-HPLC analysis were performed in the isocratic mode at 1 mL/min with column temperature at 25° C. After filtration (0.22 μm-cut-off), samples were injected at 100 μL and analyzed at 280 nm. The total run time was 35 min.

Production of Human IgE Antibodies

[0280] Anti-nitrophenyl hIgE were produced and purified as described previously (B albino B et al., The anti-IgE mAb omalizumab induces adverse reactions by engaging Fcgamma receptors. J Clin Invest. 2020). JW8/5/13 (ECACC 87080706) cells were obtained from Sigma-Aldrich. This cell line produces a chimeric human IgE antibody directed against the hapten 4-hydroxy-3-nitrophenacetyl (NP), and composed of the human Fcε chain and mouse anti-NP variable chain. JW8/5/13 cells were cultured in complete Dulbecco-modified Eagle medium (DMEM, Gibco) containing 2 mM glutamine (Thermo Fisher Scientific) and 10% Fetal Bovine Serum (FBS) (Thermo Fisher Scientific) at 9×10.sup.5 cells/ml. After 15 days, supernatants were harvested, centrifuged at 4200 rpm for 30 min and filtered (0.2 μm). We purified IgE antibodies by affinity chromatography. Briefly, CNBr-activated Sepharose 4 Fast Flow Beads (GE Healthcare) were coupled with WT anti-IgE using a ratio of 2.5 mg of protein for each gram of beads. Beads were weighted, washed with 15 volumes of cold 1 mM HCl and centrifuged for 5 min at 2500 rpm. WT anti-IgE were resuspended in coupling solution (0.1 M NaHCO3 pH 8.3 containing 0.5M NaCl) and mixed with beads overnight at 4° C. under agitation. Beads were washed with coupling buffer and non-reacted groups were blocked with 0.1 M Tris-HCl buffer pH 8.0. WT anti-IgE-coupled beads were then washed using alternate low (0.1 M acetate buffer pH 3) and high (0.1 M Tris-HCl pH 8) pH solutions and stored in Borate buffer (100 mM Borate, 150 mM NaCl pH 8.0) at 4° C. until use. For purification of IgE, WT anti-IgE-coupled sepharose beads were packed in XK 16/20 Column (GE Healthcare) and affinity chromatography was performed using an AKTA pure FPLC instrument (GE Healthcare). After purification, IgE antibodies were desalted with HiTrap Desalting Column (GE Healthcare), and stored at 4° C. until use.

Vaccination with hIgE Kinoid

[0281] Mice were immunized intramuscularly with hIgE-K combined 1:1 (v:v) with SWE, a squalene-in-water emulsion adjuvant (Vaccine Formulation Laboratory, University of Lausanne, Switzerland) in PBS at day 0, 7 and 28 with two initial doses of 30 μg followed by a boost of 10 μg. As controls, groups of mice were injected with CRM.sub.197 following the same schedule with two initial doses of 15 μg followed by a boost of 5 μg (these doses were defined based on the weight ratio of CRM.sub.197) combined with SWE.

Quantification of IgG Against Human IgE and CRM.sub.197 in Sera from Vaccinated Mice

[0282] The immunogenicity of the kinoid was assessed by evaluating antibodies against human IgE and CRM.sub.197 in sera collected at different time points after vaccination. Human IgE or CRM.sub.197 were coated at 4° C. at 5 or 1 μg/mL respectively in coating buffer (carbonate/bicarbonate buffer pH 9.6) and incubated overnight. After each step, plates were washed three times with PBS Tween 20 at 0.005%. After blocking with BSA 1% PBS, serum samples were added, a two-fold serial dilution was conducted starting at 2000 dil.sup.−1 (diluted in PBS, BSA 1%). After 90 minutes of incubation at 37° C., bound antibodies were detected with HRP-conjugated goat anti mouse IgG (Bethyl Laboratories) at 1/10 000 and plates were revealed using an OPD substrate. Reaction was stopped with 1 M H.sub.2SO.sub.4 and absorbance was subsequently recorded at 490 nm. Samples were analyzed starting at dilution 2000 dil.sup.−1 up to 1 024 000 dil.sup.−1. The titers were defined as the dilution of the serum where 50% of the OD max. Titers were expressed as serum dilution factors (dil.sup.−1). The limit of titer quantification is the lowest dilution tested in the assay: 2000 dil.sup.−1.

Assessment of the Neutralizing Capacities of Anti-hIgE Antibodies Produced Upon Vaccination with hIgE-K

[0283] Bone marrow-derived cultured mast cells (BMCMCs) expressing hFcεRI were obtained by culturing bone marrow cells from IgE/FcεRI humanized mice in medium containing IL-3 (10 ng/ml) for 6 weeks, at which time cells were >95% c-Kit.sup.+hFcεRIα.sup.+ (data not shown). To assess the neutralizing capacity of anti-hIgE antibodies produced upon vaccination with hIgE-K, we incubated BMCMCs with dilutions of plasma from mice vaccinated with hIgE-K or CRM.sub.197 alone (as a control). We then added FITC-labeled hIgE (produced as described previously in B albino B et al., The anti-IgE mAb omalizumab induces adverse reactions by engaging Fcgamma receptors. J Clin Invest. 2020), and assess binding of FITC-hIgE to hFcεRI on BCMMCs by flow cytometry.

IgE Quantification on the Surface of Basophils and Mast Cells

[0284] Blood was collected with heparin. For peritoneal lavage fluid (PLF), the outer skin of the peritoneum was gently removed. Then 3 mL of cold PBS was injected into the peritoneal cavity using a 27 g needle. After a gently massage of the peritoneum, an incision was performed in the inner skin of the peritoneum and while holding up the skin with forceps, the PLF was recovered.

Red Blood Cell Lysis was Carried Out to Remove Red Blood Cells

[0285] Cells coming from blood were stained with anti CD49b-BV421 (clone DXS, eBioscience), anti CD131-PE (clone REA193, Miltenyi) and with anti-human IgE-biotin (clone MHE-18, Biolegend) and anti-Biotin-APC (clone REA746, Miltenyi) or anti-human FcεRI-APC (clone AER-37 (CRA-1), BioLegend). Cells coming from PLF were stained with anti cKIT-APC (clone 2B8, eBioscience) and with anti-human IgE biotin (clone MHE-18, BioLegend) and anti-Biotin-APC (clone REA746, Miltenyi) or anti-human FcεRI-APC (clone AER-37 (CRA-1), BioLegend). Basophils were gated as CD49b.sup.+ CD131.sup.+ and mast cells as cKIT.sup.+IgE.sup.+ or FcεRI.sup.+. Surface expression of human FcεRI and IgE was assessed and expressed by mean fluorescence intensity (MFI).

Total Human and Mouse IgE Quantification

[0286] Total human IgE levels were quantified by ELISA. Anti-Cε2 human IgE antibody (clone 8E/5D4, Aviva Systems Biology) was coated and incubated overnight at 4° C. at 5 μg/mL in coupling buffer (carbonate/bicarbonate buffer pH=9.6). After each step, plates were washed three times with PBS Tween 20 at 0.005%. After blocking with BSA 1% in PBS for 1 h30 at room temperature, serum samples were added at 1/10 final dilution (diluted in PBS, BSA 1% 10% FBS) and incubated for 90 minutes at room temperature. Then, anti-human IgE antibody (A80-108P, Bethyl Laboratories) were added at 1:10,000 during 90 minutes at room temperature. Plates were revealed using OPD substrate. Reaction was stopped with 2 M H.sub.2SO.sub.4 and absorbance was subsequently recorded at 490 nm. Total mouse IgE levels were quantified by ELISA using a commercial ELISA kit (E90-115; B ethyl Laboratories) according to the manufacturer's instructions.

Passive Systemic Anaphylaxis

[0287] In IgE/FcεRI humanized mice, purified mouse IgE anti-NP antibodies were administered intravenously (i.v.) at a dose of 10 μg in 100 μL of PBS. Twenty-four hours later, mice were challenged i.v. with 500 μg of NP (21-31)-BSA (Santa Cruz Biotechnology) in PBS. Rectal measurements of body temperature were performed immediately before (time 0) and at different time points for up to one hour after challenge. In IgE/FcεRI humanized; F709 IL4Ra mice, rabbit anti-hIgE antibodies (Bethyl Laboratories) were administered i.v. at a dose of 250 μg. Rectal measurements of body temperature were performed immediately before (time 0) and at different time points for up to one hour after the injection.

Statistical Analysis

[0288] Statistical significance was determined using the unpaired Student's t test (unpaired Mann Whitney test). P≤0.05 was considered statistically significant. Calculations were performed using the Prism 7.0 software program (GraphPad Software).

Results

[0289] Vaccination with hIgE Kinoid Induces Potent Anti-IgE Neutralizing Antibodies in IgE Humanized Mice

[0290] IgE kinoids (hIgE-K) were generated by coupling human IgE Cε3-4 domains with diphtheria ‘cross-reactive material .sub.197’ (CRM.sub.197, a non-toxic mutant of diphtheria toxin used as a carrier protein in a number of approved conjugated vaccines) using a thiol-maleimide conjugation (FIG. 1A). We replaced the native glycine residue at position 335 by a cysteine residue into Cε3-4. Consequently, interchain disulfide bonds are formed that locks the IgE fragment into a “closed” conformation retaining high-affinity binding to omalizumab, but not FcεRI. We hypothesized that an IgE conjugate vaccine containing this G335C mutation would favor generation of “omalizumab-like” neutralizing antibodies while avoiding potentially harmful binding to FcεRI. SDS-PAGE and HPLC analysis indicated formation of high molecular species upon conjugation of hIgE Cε3-4 G335C to CRM.sub.197, confirming efficiency synthesis of hIgE-K (FIGS. 1B and 1C).

[0291] Immunization of hIgE.sup.KI mice (which express human IgE instead of mouse IgE) with hIgE-K in SWE, a squalene oil-in-water emulsion adjuvant, induced high anti-hIgE antibody titers, detectable already 5 weeks after primary immunization and still more than 39 weeks after (the latest time-point assessed so far) (FIG. 2A-B). As expected, all mice exposed to CRM.sub.197 alone or hIgE-K developed anti-CRM.sub.197 antibodies (FIG. 2C). Importantly, anti-hIgE antibodies generated upon vaccination with the kinoid exhibited strong neutralizing capacities in all mice starting 5 weeks after primary immunization (FIG. 2D). We could detect hIgE in the blood of CRM.sub.197-immunized control mice, but not in hIgE.sup.KI mice vaccinated with the hIgE-K, confirming the neutralizing capacities of antibodies generated upon vaccination (FIG. 2E). Altogether, these data indicate that efficient long-term neutralization of hIgE can be achieved through vaccination with hIgE-K in hIgE.sup.KI mice.

Efficacy of Anti-hIgE Vaccine in a Model of hIgE-Mediated Anaphylaxis

[0292] We assessed potential adverse events following injection of hIgE-K in mice expressing both human IgE and human FcεRI (IgE/FcεRI humanized mice). We carefully monitored mice after each injection of IgE-K vaccine (or CRM.sub.197 alone as a control) (FIG. 3A) and did not observe any detectable adverse effect in IgE/FcεRI humanized mice: neither hypothermia (FIG. 3C), the parameter used to follow anaphylactic shock in mice, nor diarrhea, distress or lack of vitality over 1 hour following each vaccine injection in IgE/FcεRI humanized mice. This absence of adverse effects suggests that the vaccine does not trigger FcεRI activation through IgE aggregation on the surface of mast cells and basophils. Vaccination with hIgE-K induced high titers of anti-hIgE antibodies in IgE/FcεRI humanized mice, which were already detectable 5 weeks after the first injection of kinoid (FIG. 3B), similarly to their appearance in hIgE.sup.KI mice (FIG. 2E).

[0293] To further assess the safety and efficiency of the hIgE vaccine, we injected a high dose (10 μg) of anti-nitrophenyl (NP) hIgE into IgE/FcεRI humanized mice which had been vaccinated with hIgE-K or with CRM.sub.197 alone as a control, following the same immunization schedule described above (FIG. 3A). Again, we observed neither hypothermia, nor diarrhea, distress or lack of vitality over 1 hour following injection of anti-NP-hIgE, confirming that the vaccine does not induce detectable side effects even in the presence of very high levels of circulating hIgE (FIG. 3D). Importantly, mice vaccinated with the hIgE-K were protected from hIgE-mediated anaphylaxis, whereas CRM.sub.197-vaccinated mice injected with anti-NP hIgE and challenged with the NP antigen suffered profound hypothermia and 1 out of 7 mice died (FIG. 3E).

Efficacy of Anti-hIgE Vaccine in a Genetically Predisposed Allergic Mouse Model

[0294] IgE/FcεRI humanized mice demonstrated low levels of circulating hIgE, whereas allergic patients display moderate to high levels of circulating IgE, making the mouse model potentially easier to protect from IgE-induced events following anti-IgE vaccination. To resolve this discrepancy, we crossed IgE/FcεRI humanized mice with mice bearing the gain-of-function Y709F mutation in the gene encoding the interleukin-4 (IL-4) and IL-13 receptor subunit, IL-4Ra, to generate hIgE.sup.KI; hFcεRI.sup.Tg; F709 IL4Ra mice (FIG. 4A). The Y709F mutation disrupts the Immunoreceptor tyrosine-based inhibitory motif (ITIM) of IL4Ra, thus enhancing receptor signaling in response to IL-4 and IL-13, amplifying IgE levels and IgE-mediated anaphylaxis. We used hIgE.sup.KI; hFcεRI.sup.Tg; F709 IL4Ra mice to assess the efficiency of the hIgE vaccine in a model in which anaphylaxis is triggered by endogenous hIgE. To do so, we injected hIgE.sup.KI; hFcεRI.sup.Tg; F709 IL4Ra mice with a high dose of polyclonal anti-hIgE Abs to trigger mast cell activation through crosslink of FcεRI-bound hIgE. CRM.sub.197-immunized mice, used as controls, developed severe hypothermia (FIG. 4B) with 100% mortality within 30 min after anti-hIgE injection (FIG. 4C), confirming that IgE/FcεRI humanized; F709 IL4Ra mice have sufficient levels of endogenous hIgE bound to hFcεRI to trigger hIgE-mediated anaphylaxis. By great contrast, after anti-hIgE injection, IgE-K vaccinated mice displayed only a transient mild hypothermia and suffered no mortality (FIG. 4B-C).

[0295] Altogether, our results indicate that a vaccine against human IgE Cε3-4 domains can be produced using standard industrial methods, and this vaccine can lead to long-term neutralization of hIgE leading to undetectable IgE levels in circulation and reduced FcεRI-bound hIgE. hIgE-K vaccination does not induce any detectable adverse effects in mice humanized for IgE and FcεRI, even after repeated injections. IgE-K vaccination leads to protection from severe IgE-mediated allergic reactions, even in genetically predisposed allergic mouse models. These results pave the way for the clinical development of an efficient long-term vaccine against hIgE-mediated allergic disorders.