Targeting inhibitory Siglec-3 to suppress IgE-mediated human basophil degranulation.

BACKGROUND: Sialic acid-binding immunoglobulin-like lectin-3 (Siglec-3 [CD33]) is a major Siglec expressed on human mast cells and basophils; engagement of CD33 leads to inhibition of cellular signaling via immunoreceptor tyrosine-based inhibitory motifs. OBJECTIVE: We sought to inhibit human basophil degranulation by simultaneously recruiting inhibitory CD33 to the IgE-FcεRI complex by using monoclonal anti-IgE directly conjugated to CD33 ligand (CD33L). METHODS: Direct and indirect basophil activation tests (BATs) were used to assess both antigen-specific (peanut) and antigen-nonspecific (polyclonal anti-IgE) stimulation. Whole blood from donors with allergy was used for direct BAT, whereas blood from donors with nonfood allergy was passively sensitized with plasma from donors with peanut allergy in the indirect BAT. Blood was incubated with anti-IgE-CD33L or controls for 1 hour or overnight and then stimulated with peanut, polyclonal anti-IgE, or N-formylmethionyl-leucyl-phenylalanine for 30 minutes. Degranulation was determined by measuring CD63 expression on the basophil surface by flow cytometry. RESULTS: Incubation for 1 hour with anti-IgE-CD33L significantly reduced basophil degranulation after both allergen-induced (peanut) and polyclonal anti-IgE stimulation, with further suppression after overnight incubation with anti-IgE-CD33L. As expected, anti-IgE-CD33L did not block basophil degranulation due to N-formylmethionyl-leucyl-phenylalanine, providing evidence that this inhibition is IgE pathway-specific. Finally, CD33L is necessary for this suppression, as monoclonal anti-IgE without CD33L was unable to reduce basophil degranulation. CONCLUSIONS: Pretreating human basophils with anti-IgE-CD33L significantly suppressed basophil degranulation through the IgE-FcεRI complex. The ability to abrogate IgE-mediated basophil degranulation is of particular interest, as treatment with anti-IgE-CD33L before antigen exposure could have broad implications for the treatment of food, drug, and environmental allergies.

A mutation in Themis contributes to anaphylaxis severity following oral peanut challenge in CC027 mice.

BACKGROUND: The development of peanut allergy is due to a combination of genetic and environmental factors, although specific genes have proven difficult to identify. Previously, we reported that peanut-sensitized Collaborative Cross strain CC027/GeniUnc (CC027) mice develop anaphylaxis upon oral challenge to peanut, in contrast to C3H/HeJ (C3H) mice. OBJECTIVE: This study aimed to determine the genetic basis of orally induced anaphylaxis to peanut in CC027 mice. METHODS: A genetic mapping population between CC027 and C3H mice was designed to identify the genetic factors that drive oral anaphylaxis. A total of 356 CC027xC3H backcrossed mice were generated, sensitized to peanut, then challenged to peanut by oral gavage. Anaphylaxis and peanut-specific IgE were quantified for all mice. T-cell phenotyping was conducted on CC027 mice and 5 additional Collaborative Cross strains. RESULTS: Anaphylaxis to peanut was absent in 77% of backcrossed mice, with 19% showing moderate anaphylaxis and 4% having severe anaphylaxis. There were 8 genetic loci associated with variation in response to peanut challenge-6 associated with anaphylaxis (temperature decrease) and 2 associated with peanut-specific IgE levels. There were 2 major loci that impacted multiple aspects of the severity of acute anaphylaxis, at which the CC027 allele was associated with worse outcome. At one of these loci, CC027 has a private genetic variant in the Themis gene. Consistent with described functions of Themis, we found that CC027 mice have more immature T cells with fewer CD8+, CD4+, and CD4+CD25+CD127- regulatory T cells. CONCLUSIONS: Our results demonstrate a key role for Themis in the orally reactive CC027 mouse model of peanut allergy.

Desensitization and remission after peanut sublingual immunotherapy in 1- to 4-year-old peanut-allergic children: A randomized, placebo-controlled trial.

BACKGROUND: Prior studies of peanut sublingual immunotherapy (SLIT) have suggested a potential advantage with younger age at treatment initiation. OBJECTIVE: We studied the safety and efficacy of SLIT for peanut allergy in 1- to 4-year-old children. METHODS: Peanut-allergic 1- to 4-year-old children were randomized to receive 4 mg peanut SLIT versus placebo. Desensitization was assessed by double-blind, placebo-controlled food challenge (DBPCFC) after 36 months of treatment. Participants desensitized to at least 443 mg peanut protein discontinued therapy for 3 months and then underwent DBPCFC to assess for remission. Biomarkers were measured at baseline and longitudinally during treatment. RESULTS: Fifty participants (25 peanut SLIT, 25 placebo) with a median age of 2.4 years were enrolled across 2 sites. The primary end point of desensitization was met with actively treated versus placebo participants having a significantly greater median cumulative tolerated dose (4443 mg vs 143 mg), higher likelihood of passing the month 36 DBPCFC (60% vs 0), and higher likelihood of demonstrating remission (48% vs 0). The highest rate of desensitization and remission was seen in 1- to 2-year-olds, followed by 2- to 3-year-olds and 3- to 4-year-olds. Longitudinal changes in peanut skin prick testing, peanut-specific IgG4, and peanut-specific IgG4/IgE ratio were seen in peanut SLIT but not placebo participants. Oropharyngeal itching was more commonly reported by peanut SLIT than placebo participants. Skin, gastrointestinal, upper respiratory, lower respiratory, and multisystem adverse events were similar between treatment groups. CONCLUSION: Peanut SLIT safely induces desensitization and remission in 1- to 4-year-old children, with improved outcomes seen with younger age at initiation.

Different airborne particulates trigger distinct immune pathways leading to peanut allergy in a mouse model.

BACKGROUND: Environmental exposure to peanut through non-oral routes is a risk factor for peanut allergy. Early-life exposure to air pollutants, including particulate matter (PM), is associated with sensitization to foods through unknown mechanisms. We investigated whether PM promotes sensitization to environmental peanut and the development of peanut allergy in a mouse model. METHODS: C57BL/6J mice were co-exposed to peanut and either urban particulate matter (UPM) or diesel exhaust particles (DEP) via the airways and assessed for peanut sensitization and development of anaphylaxis following peanut challenge. Peanut-specific CD4+ T helper (Th) cell responses were characterized by flow cytometry and Th cytokine production. Mice lacking select innate immune signaling genes were used to study mechanisms of PM-induced peanut allergy. RESULTS: Airway co-exposure to peanut and either UPM- or DEP-induced systemic sensitization to peanut and anaphylaxis following peanut challenge. Exposure to UPM or DEP triggered activation and migration of lung dendritic cells to draining lymph nodes and induction of peanut-specific CD4+ Th cells. UPM- and DEP-induced distinct Th responses, but both stimulated expansion of T follicular helper (Tfh) cells essential for peanut allergy development. MyD88 signaling was critical for UPM- and DEP-induced peanut allergy, whereas TLR4 signaling was dispensable. DEP-induced peanut allergy and Tfh-cell differentiation depended on IL-1 but not IL-33 signaling, whereas neither cytokine alone was necessary for UPM-mediated sensitization. CONCLUSION: Environmental co-exposure to peanut and PM induces peanut-specific Tfh cells and peanut allergy in mice.

IgE and IgG4 epitopes of the peanut allergens shift following oral immunotherapy.

Background: Oral immunotherapy (OIT) with peanut (Arachis hypogaea) allergen powder-dnfp (PTAH; Aimmune Therapeutics) is an FDA-approved treatment to desensitize peanut allergic participants. Objective: Here we assessed shifts in IgE and IgG4 binding to peanut allergens and their epitopes recognized by United States (US) peanut allergic participants (n = 20) enrolled in phase 3 PTAH OIT clinical trials. Methods: Pre- and post- trial participant sera were collected approximately 12 months apart and tested for IgE binding to intact peanut proteins via ImmunoCAP ISAC immunoassays. IgE and IgG4 linear epitopes were identified based on binding to synthetic overlapping 15-mer linear peptides of 10 peanut allergens (Ara h 1-11) synthesized on microarray slides. Results: Statistically significant decreases in IgE binding were identified for intact Ara h 2, 3, and 6, and known and newly identified IgE epitopes were shown to exhibit shifts towards IgG4 binding post-OIT, with most linear peptides having increased IgG4 binding after treatment with PTAH. While PTAH does not seem to alter the actual peptide binding patterns significantly after one year of treatment, the IgE and IgG4 binding ratios and intensity are altered. Conclusion: At a population level, the linear IgE and IgG4 epitopes of 10 peanut allergens overlap and that increase in IgG4 with OIT results in displacement of IgE binding to both conformational and linear epitopes. Furthermore, it appears as though the increase in IgG4 is more important to achieve desensitization at the 12-month timepoint than the decrease in IgE. This type of knowledge can be useful in the identification of IgE and IgG4-binding allergen and peptide biomarkers that may indicate desensitization or sustained unresponsiveness of allergic individuals to peanut.

Alanine Scanning of the Unstructured Region of Ara h 2 and of a Related Mimotope Reveals Critical Amino Acids for IgE Binding.

SCOPE: The unstructured region of Ara h 2, referred to as epitope 3, contains a repeated motif, DYPSh (h = hydroxyproline) that is important for IgE binding. METHODS AND RESULTS: IgE binding assays to 20mer and shorter peptides of epitope 3, defines a 16mer core sequence containing one copy of the DPYSh motif, DEDSYERDPYShSQDP. This study performs alanine scanning of this and a related 12mer mimotope, LLDPYAhRAWTK. IgE binding, using a pool of 10 sera and with individual sera, is greatly reduced when alanine is substituted for aspartate at position 8 (D8; p < 0.01), tyrosine at position 10 (Y10; p < 0.01), and hydroxyproline at position 12 (h12; p < 0.001). IgE binding to alanine-substituted peptides of a mimotope containing the DPY_h motif confirm the critical importance of Y (p < 0.01) and h (p < 0.01), but not D. Molecular modeling of the core and mimotope suggests an h-dependent conformational basis for the recognition of these sequences by polyclonal IgE. CONCLUSIONS: IgE from pooled sera and individual sera differentially bound amino acids throughout the sequences of Epitope 3 and its mimotope, with Y10 and h12 being most important for all sera. These results are highly significant for designing hypoallergenic forms of Ara h 2.

A mutation in Themis contributes to peanut-induced oral anaphylaxis in CC027 mice.

Background: The development of peanut allergy is due to a combination of genetic and environmental factors, although specific genes have proven difficult to identify. Previously, we reported that peanut-sensitized CC027/GeniUnc (CC027) mice develop anaphylaxis upon oral challenge to peanut, unlike C3H/HeJ (C3H) mice. Objective: To determine the genetic basis of orally-induced anaphylaxis to peanut in CC027 mice. Methods: A genetic mapping population between CC027 and C3H mice was designed to identify the genetic factors that drive oral anaphylaxis. A total of 356 CC027xC3H backcrossed mice were generated, sensitized to peanut, then challenged to peanut by oral gavage. Anaphylaxis and peanut-specific IgE were quantified for all mice. T-cell phenotyping was conducted on CC027 and five additional CC strains. Results: Anaphylaxis to peanut was absent in 77% of backcrossed mice, with 19% showing moderate anaphylaxis, and 4% having severe anaphylaxis. A total of eight genetic loci were associated with variation in response to peanut challenge, six associated with anaphylaxis (temperature decrease) and two associated with peanut-specific IgE levels. There were two major loci that impacted multiple aspects of the severity of acute anaphylaxis, at which the CC027 allele was associated with worse outcome. At one of these loci, CC027 has a private genetic variant in the Themis (thymocyte-expressed molecule involved in selection) gene. Consistent with Themis' described functions, we found that CC027 have more immature T cells with fewer CD8+, CD4+, and CD4+CD25+CD127- regulatory T cells. Conclusion: Our results demonstrate a key role for Themis in the orally-reactive CC027 mouse model of peanut allergy.

Food-specific IgA levels in esophageal biopsies are not sufficiently high to predict food triggers in eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is an immune-mediated disease, characterized by Th2-type inflammation linked to specific foods. No currently available allergy tests reliably identify food triggers in EoE, leading to empiric dietary elimination strategies. Recently, milk- and wheat-specific IgA in esophageal brushings were linked to clinical food triggers. In this study, we aimed to determine whether food-specific IgA from esophageal biopsies is associated with known food triggers. METHODS: A prior cohort of 21 patients (median age 39 years) with confirmed EoE underwent empirical elimination diets and subsequent reintroduction of foods to determine triggers. Archived baseline biopsies were used to quantify levels of peanut-, milk-, soy-, egg-, wheat-specific and total IgA by enzyme-linked immunosorbent assay. RESULTS: Overall, 13 patients (62%) responded to the dietary elimination as determined by histology (<15 eos/hpf), with milk and egg being the most common triggers. Biopsies had varying amounts of total IgA, while food-specific IgA was only detectable in 48 of 105 (46%) samples. Food-specific IgA was normalized to total IgA for each sample and stratified by whether a food was a known trigger. For all foods tested, there were no significant differences in IgA between positive and negative triggers. CONCLUSIONS: Food-specific IgA in esophageal biopsies was not associated with previously identified food triggers in this cohort. Future studies comparing food-specific IgA in esophageal brushings, mucous scrapings, and biopsies from patients with known triggers will be critical to determining whether food-specific IgA may serve as a biomarker for identification of EoE triggers.

Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice.

Background: Early dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains. Objective: We aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains. Methods: C57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed. Results: Without oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge. Conclusion: Allergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate.

A single priming event prevents oral tolerance to peanut.

BACKGROUND: Indoor dust (ID) is a source of peanut proteins and immunostimulatory adjuvants (e.g. LPS) that can promote airway sensitization to peanut. We aimed to determine whether a single airway exposure to peanut plus adjuvant is sufficient to prevent oral tolerance. METHODS: To determine the effect of a single priming event, C57BL/6J mice were exposed once to peanut plus adjuvant through the airway, followed by either airway or low-dose oral exposure to peanut, and assessed for peanut allergy. Oral tolerance was investigated by feeding high-dose peanut followed by airway sensitization. To determine whether a single priming could prevent oral tolerance, the high-dose peanut regimen was applied after a single airway exposure to peanut plus adjuvant. Peanut-specific IgE and IgG1 were quantified, and mice were challenged to peanut to assess allergy. Peanut-specific CD4+ memory T cells (CD4+ TCRß+ CD44hi CD154+ ) were quantified in mediastinal lymph nodes following airway priming. RESULTS: Mice co-exposed to peanut with LPS or ID through the airway were primed to develop peanut allergy after subsequent low-dose oral or airway exposures to peanut. Oral tolerance was induced in mice fed high-dose peanut prior to airway sensitization. In contrast, mice fed high-dose peanut following a single airway exposure to peanut plus adjuvant led to allergy. Peanut-specific CD4+ memory T cells were detected as early as 7 days after the single airway priming with peanut plus adjuvant, however, delaying peanut feeding even 1 day following priming led to allergy, whereas peanut feeding the same day as priming led to tolerance. CONCLUSIONS: A single airway exposure to peanut plus adjuvant is sufficient to prime the immune system to develop allergy following subsequent high-dose oral exposure. These results highlight the importance of introducing peanut as early as possible to prevent sensitization through a non-oral priming event.

Open-label study of the efficacy, safety, and durability of peanut sublingual immunotherapy in peanut-allergic children.

BACKGROUND: Studies on the efficacy of peanut sublingual immunotherapy (SLIT) are limited. The durability of desensitization after SLIT has not been well described. OBJECTIVE: We sought to evaluate the efficacy and safety of 4-mg peanut SLIT and persistence of desensitization after SLIT discontinuation. METHODS: Challenge-proven peanut-allergic 1- to 11-year-old children were treated with open-label 4-mg peanut SLIT for 48 months. Desensitization after peanut SLIT was assessed by a 5000-mg double-blind, placebo-controlled food challenge (DBPCFC). A novel randomly assigned avoidance period of 1 to 17 weeks was followed by the DBPCFC. Skin prick test results immunoglobulin levels, basophil activation test results, TH1, TH2, and IL-10 cytokines were measured longitudinally. Safety was assessed through patient-reported home diaries. RESULTS: Fifty-four participants were enrolled and 47 (87%) completed peanut SLIT and the 48-month DBPCFC per protocol. The mean successfully consumed dose (SCD) during the DBPCFC increased from 48 to 2723 mg of peanut protein after SLIT (P < .0001), with 70% achieving clinically significant desensitization (SCD > 800 mg) and 36% achieving full desensitization (SCD = 5000 mg). Modeled median time to loss of clinically significant desensitization was 22 weeks. Peanut skin prick test; peanut-specific IgE, IgG4, and IgG4/IgE ratio; and peanut-stimulated basophil activation test, IL-4, IL-5, IL-13, IFN-γ, and IL-10 changed significantly compared with baseline, with changes seen as early as 6 months. Median rate of reaction per dose was 0.5%, with transient oropharyngeal itching being the most common, and there were no dosing symptoms requiring epinephrine. CONCLUSIONS: In this open-label, prospective study, peanut SLIT was safe and induced clinically significant desensitization in most of the children, lasting more than 17 weeks after discontinuation of therapy.

Structure and IgE Cross-Reactivity among Cashew, Pistachio, Walnut, and Peanut Vicilin-Buried Peptides.

Peanut and tree-nut allergies are frequently comorbid for reasons not completely understood. Vicilin-buried peptides (VBPs) are an emerging family of food allergens whose conserved structural fold could mediate peanut/tree-nut co-allergy. Peptide microarrays were used to identify immunoglobulin E (IgE) epitopes from the N-terminus of the vicilin allergens Ara h 1, Ana o 1, Jug r 2, and Pis v 3 using serum from three patient diagnosis groups: monoallergic to either peanuts or cashew/pistachio, or dual allergic. IgE binding peptides were highly prevalent in the VBP domains AH1.1, AO1.1, JR2.1, and PV3.1, but not in AO1.2, JR2.2, JR2.3, and PV3.2 nor the unstructured regions. The IgE profiles did not correlate with diagnosis group. The structure of the VBPs from cashew and pistachio was solved using solution-NMR. Comparisons of structural features suggest that the VBP scaffold from peanuts and tree-nuts can support cross-reactivity. This may help understand comorbidity and cross-reactivity despite a distant evolutionary origin.

Manipulating the microbiome to enhance oral tolerance in food allergy.

Loss of oral tolerance (OT) to food antigens results in food allergies. One component of achieving OT is the symbiotic microorganisms living in the gut (microbiota). The composition of the microbiota can drive either pro-tolerogenic or pro-inflammatory responses against dietary antigens though interactions with the local immune cells within the gut. Products from bacterial fermentation, such as butyrate, are one of the main communication molecules involved in this interaction, however, this is released by a subset of bacterial species. Thus, strategies to specifically expand these bacteria with protolerogenic properties have been explored to complement oral immunotherapy in food allergy. These approaches either provide digestible biomolecules to induce beneficial bacteria species (prebiotics) or the direct administration of live bacteria species (probiotics). While this combined therapy has shown positive outcomes in clinical trials for cow's milk allergy, more research is needed to determine if this therapy can be extended to other food allergens.

Food-specific immunoglobulin A does not correlate with natural tolerance to peanut or egg allergens.

ImmunoglobulinA (IgA) is the predominant antibody isotype in the gut, where it regulates commensal flora and neutralizes toxins and pathogens. The function of food-specific IgA in the gut is unknown but is presumed to protect from food allergy. Specifically, it has been hypothesized that food-specific IgA binds ingested allergens and promotes tolerance by immune exclusion; however, the evidence to support this hypothesis is indirect and mixed. Although it is known that healthy adults have peanut-specific IgA in the gut, it is unclear whether children also have gut peanut-specific IgA. We found in a cohort of non-food-allergic infants (n = 112) that there is detectable stool peanut-specific IgA that is similar to adult quantities of gut peanut-specific IgA. To investigate whether this peanut-specific IgA is associated with peanut tolerance, we examined a separate cohort of atopic children (n = 441) and found that gut peanut-specific IgA does not predict protection from development of future peanut allergy in infants nor does it correlate with concurrent oral tolerance of peanut in older children. We observed higher plasma peanut-specific IgA in those with peanut allergy. Similarly, egg white-specific IgA was detectable in infant stools and did not predict egg tolerance or outgrowth of egg allergy. Bead-based epitope assay analysis of gut peanut-specific IgA revealed similar epitope specificity between children with peanut allergy and those without; however, gut peanut-specific IgA and plasma peanut-specific IgE had different epitope specificities. These findings call into question the presumed protective role of food-specific IgA in food allergy.

Induction of food-specific IgG by Gene Gun-delivered DNA vaccines.

Background: Shellfish and tree nut allergies are among the most prevalent food allergies, now affecting 2%-3% and 1% of the US population, respectively. Currently, there are no approved therapies for shellfish or tree nut allergies, with strict avoidance being the standard of care. However, oral immunotherapy for peanut allergy and subcutaneous immunotherapy for environmental allergens are efficacious and lead to the production of allergen-specific IgG, which causes suppression of allergen effector cell degranulation. Since allergen-specific IgG is a desired response to alleviate IgE-mediated allergies, we tested transcutaneously-delivered DNA vaccines targeting shellfish and tree nut allergens for their ability to induce antigen-specific IgG, which would have therapeutic potential for food allergies. Methods: We assessed Gene Gun-delivered DNA vaccines targeting either crustacean shellfish or walnut/pecan allergens, with or without IL-12, in naïve mice. Three strains of mice, BALB/cJ, C3H/HeJ and CC027/GeniUnc, were evaluated for IgG production following vaccination. Vaccines were administered twice via Gene Gun, three weeks apart and then blood was collected three weeks following the final vaccination. Results: Vaccination with shellfish allergen DNA led to increased shrimp-specific IgG in all three strains, with the highest production in C3H/HeJ from the vaccine alone, whereas the vaccine with IL-12 led to the highest IgG production in BALB/cJ and CC027/GeniUnc mice. Similar IgG production was also induced against lobster and crab allergens. For walnut/pecan vaccines, BALB/cJ and C3H/HeJ mice produced significantly higher walnut- and pecan-specific IgG with the vaccine alone compared to the vaccine with IL-12, while the CC027 mice made significantly higher IgG with the addition of IL-12. Notably, intramuscular administration of the vaccines did not lead to increased antigen-specific IgG production, indicating that Gene Gun administration is a superior delivery modality. Conclusions: Overall, these data demonstrate the utility of DNA vaccines against two lifelong food allergies, shellfish and tree nuts, suggesting their potential as a food allergy therapy in the future.

Novel peanut-specific human IgE monoclonal antibodies enable screens for inhibitors of the effector phase in food allergy.

Background: 10% of US residents have food allergies, including 2% with peanut allergy. Mast cell mediators released during the allergy effector phase drive allergic reactions. Therefore, targeting sensitized mast cells may prevent food allergy symptoms. Objective: We used novel, human, allergen-specific, IgE monoclonal antibodies (mAbs) created using human hybridoma techniques to design an in vitro system to evaluate potential therapeutics targeting sensitized effector cells. Methods: Two human IgE mAbs specific for peanut, generated through human hybridoma techniques, were used to sensitize rat basophilic leukemia (RBL) SX-38 cells expressing the human IgE receptor (FcϵRI). Beta-hexosaminidase release (a marker of degranulation), cytokine production, and phosphorylation of signal transduction proteins downstream of FcϵRI were measured after stimulation with peanut. Degranulation was also measured after engaging inhibitory receptors CD300a and Siglec-8. Results: Peanut-specific human IgE mAbs bound FcϵRI, triggering degranulation after stimulation with peanut in RBL SX-38 cells. Sensitized RBL SX-38 cells stimulated with peanut increased levels of phosphorylated SYK and ERK, signal transduction proteins downstream of FcϵRI. Engaging inhibitory cell surface receptors CD300a or Siglec-8 blunted peanut-specific activation. Conclusion: Allergen-specific human IgE mAbs, expressed from human hybridomas and specific for a clinically relevant food allergen, passively sensitize allergy effector cells central to the in vitro models of the effector phase of food allergy. Peanut reproducibly activates and induces degranulation of RBL SX-38 cells sensitized with peanut-specific human IgE mAbs. This system provides a unique screening tool to assess the efficacy of therapeutics that target allergy effector cells and inhibit food allergen-induced effector cell activation.

Single-dose AAV vector gene immunotherapy to treat food allergy.

Immunotherapies for patients with food allergy have shown some success in limiting allergic responses. However, these approaches require lengthy protocols with repeated allergen dosing and patients can relapse following discontinuation of treatment. The purpose of this study was to test if a single dose of an adeno-associated virus (AAV) vector can safely prevent and treat egg allergy in a mouse model. AAV vectors expressing ovalbumin (OVA) under an ubiquitous or liver-specific promoter were injected prior to or after epicutaneous sensitization with OVA. Mice treated with either AAV8-OVA vector were completely protected from allergy sensitization. These animals had a significant reduction in anaphylaxis mediated by a reduction in OVA-specific IgE titers. In mice with established OVA allergy, allergic responses were mitigated only in mice treated with an AAV8-OVA vector expressing OVA from an ubiquitous promoter. In conclusion, an AAV vector with a liver-specific promoter was more effective for allergy prevention, but higher OVA levels were necessary for reducing symptoms in preexisting allergy. Overall, our AAV gene immunotherapy resulted in an expansion of OVA-specific FoxP3+ CD4+ T cells, an increase in the regulatory cytokine IL-10, and a reduction in the IgE promoting cytokine IL-13.

Peanut-Specific IgG4 and IgA in Saliva Are Modulated by Peanut Oral Immunotherapy.

BACKGROUND: Antigen-specific immunoglobulin responses have yet to be studied at the oral mucosal surface during peanut oral immunotherapy (PnOIT). OBJECTIVE: We aimed to quantify salivary peanut-specific IgG4 (PNsIgG4) and IgA (PNsIgA) and total IgG4 and IgA in participants from the Immune Tolerance Network's IMPACT study, a phase 2 PnOIT trial. METHODS: Peanut-allergic children, aged 12 months to younger than 48 months at screening, were enrolled and randomized to PnOIT or placebo oral immunotherapy (OIT) for 134 weeks. Per-protocol analysis included 69 PnOIT and 23 placebo participants. Double-blind, placebo-controlled food challenges were conducted at weeks 134 and 160 to assess desensitization and remission, respectively. Saliva samples were collected at baseline and 30, 82, 134, and 160 weeks to quantify PNsIgG4, PNsIgA, and total IgG4 and IgA. RESULTS: Participants who received PnOIT experienced significant increases in PNsIgG4 in saliva, whereas participants on placebo did not (P < .01 at all time points). The PNsIgA/total IgA ratio was also significantly increased in participants treated with PnOIT when compared with those receiving placebo at 30 and 82 weeks (P < .05). During PnOIT, desensitized participants had increased PNsIgA that plateaued, whereas the not desensitized/no remission group did not change over time. Interestingly, when the PnOIT group was evaluated by clinical outcome, PNsIgA was higher at baseline in the not desensitized/no remission group than in the desensitized/remission group (P < .05). CONCLUSIONS: PnOIT induces substantial increases in allergen-specific IgG4 and IgA in saliva. These data provide insight into OIT-induced mucosal responses and suggest the utility of these easily obtained samples for biomarker development.

Targeting CD22 on memory B cells to induce tolerance to peanut allergens.

BACKGROUND: Circulating IgE and subsequent severe allergic reactions to peanut are sustained and propagated by recall of peanut allergen-specific memory B cells. OBJECTIVES: This study aimed to determine whether targeting mouse and human CD22 on peanut-specific memory B cells induces tolerance to peanut allergens. METHODS: Siglec-engaging tolerance-inducing antigenic liposomes (STALs) codisplaying peanut allergens (Ara h 1, Ara h 2, or Ara h 3) and high-affinity CD22 ligand (CD22L-STALs) were employed in various mouse models (BALB/cJ, C57BL/6, human CD22 transgenic, and NSG) of peanut allergy. To investigate memory B cells, a conferred memory model was used in which splenocytes from peanut-sensitized mice were transferred into naive animals. Reconstituted mice received either CD22L-STALs or an immunogenic liposome control, followed by a peanut allergen boost and later a challenge with individual peanut allergens. To assess the effects of CD22L-STALs on human B cells, PBMCs were injected into NSG mice, followed by administration of human CD22L-STALs (hCD22L-STALs) and later a whole peanut extract boost. Blood was collected to quantify WPE- and Ara h 1-, 2-, and 3-specific immunoglobulins. RESULTS: Mouse CD22L-STALs (mCD22L-STALs) significantly suppressed systemic memory to Ara h 1, Ara h 2, and Ara h 3 in BALB/cJ and C57BL/6 mice, as demonstrated by reduced allergen-specific IgE, IgG1, and anaphylaxis on challenge. Importantly, 2 doses of mCD22L-STALs led to prolonged tolerance for at least 3 months. hCD22L-STALs displayed similar suppression in mice expressing human CD22 on B cells. Finally, human B cells were tolerized in vivo in NSG mice by hCD22L-STALs. CONCLUSIONS: Antigen-specific exploitation of CD22 on memory B cells can induce systemic immune tolerance.