BACKGROUND:The IMPACT trial (NCT01867671) demonstrated strong desensitization and the potential for remission with peanut oral immunotherapy (pnOIT) in 1- to 3-year-olds. Data on long-term outcomes of early intervention oral immunotherapy (OIT) are limited. OBJECTIVE:IMPACT-PLuS sought to assess the long-term efficacy, safety, and mechanistic changes related to early-life pnOIT. METHODS:Participants randomized in IMPACT (n = 146) were recruited. The primary outcome was long-term efficacy, defined as ongoing peanut consumption. Secondary outcomes included safety, peanut serology, and skin prick tests. Participants were categorized according to IMPACT treatment (pnOIT, placebo) and participation in any additional peanut allergy intervention apart from guidance given at the end of the IMPACT trial. Patients were grouped as follows: group A, pnOIT with no subsequent intervention; group B, pnOIT with subsequent intervention; group C, placebo OIT with no subsequent intervention; and group D, placebo OIT with subsequent intervention. RESULTS:Follow-up data were available for 78 of the 146 IMPACT participants (aged 9-14 years; 8-11 years after IMPACT enrollment). Fifty-eight received pnOIT in IMPACT. Overall, 80% (32/40) of group A were eating peanut at follow-up (48/58, 83%, of the entire IMPACT follow-up pnOIT group), with 35% (14/40) eating ≥1000 mg peanut. All 15 subjects from the IMPACT remission group were eating peanut at follow-up. Peanut reactions were reported by 35% (14/40) in group A, with epinephrine therapy received by 5. Compared with group C (placebo), group A had significantly lower levels of peanut and Ara h 2 IgE, and higher peanut and Ara h 2 IgG4. CONCLUSIONS:pnOIT initiated early in life can have long-term, sustainable impact, both clinically and immunologically.

Background:Food allergen immunotherapy can induce remission of food allergies in certain individuals, but the mechanisms underlying this remission are largely unknown. Prior work has identified differences in immunomodulatory metabolites between older children who develop remission versus non-remission on oral immunotherapy (OIT). Here we aim to characterize metabolomic changes during OIT in young children to and validate patterns of immunomodulatory metabolites previously observed. Methods:Untargeted plasma metabolomic profiling was performed on samples from the DEVIL peanut OIT trial (n=41, ages 9-36 months). Remission status was determined by oral food challenges conducted at the end-of-therapy and the end of a 1-month avoidance period. Linear and logistic regression models were used to detect differences in individual metabolites over time on OIT and by remission status. Pathway analyses were used to determine enrichment of chemical subclasses and biological pathways. These pathways were then compared to prior findings generated from similar profiling performed from the PNOIT peanut OIT trial (n=20, ages 7-13). Results:During OIT, glycerophospholipid metabolites (q=3.8×10 -5 ) increased over time and most amino acid metabolites (q=6.1×10 -45 ) decreased over time. Participants who went on to develop remission, had higher levels of amino acids (q=4.3×10 -8 ) and bile acids (q=0.00014), whereas children who developed non-remission had higher levels of glycerophospholipids (q=4.3×10 -10 ). Comparison of these findings with our second cohort of OIT in older children, showed replication of the enrichment of glycerophosphocholines (q=1.0×10 -13 ) and amino acids (q=2.1×10 -5 ) among metabolites that changed over time on OIT and replication of glycerophospholipids(q=5.7×10 -16 ), amino acids ( PNOIT q=7.2×10 - 7 ), and bile acids ( PNOIT q=3.8×10 -8 ) among metabolites that varied by remission status. Conclusions:Metabolomic profiles on OIT differed both over time on OIT and by OIT remission status in young children. Between two independent OIT cohorts of different ages we observed replication of significantly enriched chemical subclasses of glycerophospholipids, amino acids, and bile acids. Given the potentially immunomodulatory roles of some of these metabolites, our results suggest that glycerophospholipids, amino acids, and bile acids may be involved in the mechanisms of remission on OIT.

BACKGROUND:For young children with peanut allergy, dietary avoidance is the current standard of care. We aimed to assess whether peanut oral immunotherapy can induce desensitisation (an increased allergic reaction threshold while on therapy) or remission (a state of non-responsiveness after discontinuation of immunotherapy) in this population. METHODS:We did a randomised, double-blind, placebo-controlled study in five US academic medical centres. Eligible participants were children aged 12 to younger than 48 months who were reactive to 500 mg or less of peanut protein during a double-blind, placebo-controlled food challenge (DBPCFC). Participants were randomly assigned by use of a computer, in a 2:1 allocation ratio, to receive peanut oral immunotherapy or placebo for 134 weeks (2000 mg peanut protein per day) followed by 26 weeks of avoidance, with participants and study staff and investigators masked to group treatment assignment. The primary outcome was desensitisation at the end of treatment (week 134), and remission after avoidance (week 160), as the key secondary outcome, were assessed by DBPCFC to 5000 mg in the intention-to-treat population. Safety and immunological parameters were assessed in the same population. This trial is registered on ClinicalTrials.gov, NCT03345160. FINDINGS:Between Aug 13, 2013, and Oct 1, 2015, 146 children, with a median age of 39·3 months (IQR 30·8-44·7), were randomly assigned to receive peanut oral immunotherapy (96 participants) or placebo (50 participants). At week 134, 68 (71%, 95% CI 61-80) of 96 participants who received peanut oral immunotherapy compared with one (2%, 0·05-11) of 50 who received placebo met the primary outcome of desensitisation (risk difference [RD] 69%, 95% CI 59-79; p<0·0001). The median cumulative tolerated dose during the week 134 DBPCFC was 5005 mg (IQR 3755-5005) for peanut oral immunotherapy versus 5 mg (0-105) for placebo (p<0·0001). After avoidance, 20 (21%, 95% CI 13-30) of 96 participants receiving peanut oral immunotherapy compared with one (2%, 0·05-11) of 50 receiving placebo met remission criteria (RD 19%, 95% CI 10-28; p=0·0021). The median cumulative tolerated dose during the week 160 DBPCFC was 755 mg (IQR 0-2755) for peanut oral immunotherapy and 0 mg (0-55) for placebo (p<0·0001). A significant proportion of participants receiving peanut oral immunotherapy who passed the 5000 mg DBPCFC at week 134 could no longer tolerate 5000 mg at week 160 (p<0·001). The participant receiving placebo who was desensitised at week 134 also achieved remission at week 160. Compared with placebo, peanut oral immunotherapy decreased peanut-specific and Ara h2-specific IgE, skin prick test, and basophil activation, and increased peanut-specific and Ara h2-specific IgG4 at weeks 134 and 160. By use of multivariable regression analysis of participants receiving peanut oral immunotherapy, younger age and lower baseline peanut-specific IgE was predictive of remission. Most participants (98% with peanut oral immunotherapy vs 80% with placebo) had at least one oral immunotherapy dosing reaction, predominantly mild to moderate and occurring more frequently in participants receiving peanut oral immunotherapy. 35 oral immunotherapy dosing events with moderate symptoms were treated with epinephrine in 21 participants receiving peanut oral immunotherapy. INTERPRETATION:In children with a peanut allergy, initiation of peanut oral immunotherapy before age 4 years was associated with an increase in both desensitisation and remission. Development of remission correlated with immunological biomarkers. The outcomes suggest a window of opportunity at a young age for intervention to induce remission of peanut allergy. FUNDING:National Institute of Allergy and Infectious Disease, Immune Tolerance Network.

Despite the prevalence and severe nature of food allergy, mechanisms underlying sensitization remain to be elucidated. Previously, we demonstrated CC027/GeniUnc mice, but not C3H/HeJ mice, develop peanut allergy after oral exposure to peanut in the absence of a Th2-skewing adjuvant. Here we investigated factors contributing to sensitization following oral exposure to peanut, walnut, milk, or egg. Female CC027/GeniUnc and C3H/HeJ mice aged 4-6 weeks were sensitized weekly to peanut, walnut, milk, or egg via oral gavage for four weeks. The following week, mice were challenged to the corresponding food vial oral gavage, and body temperatures were measured to monitor anaphylaxis. Serum was collected to measure allergen-specific immunoglobulins, and fecal pellets were collected to quantify fecal IgA and analyze the gut microbiome. Naïve CC027/GeniUnc mice had markedly lower fecal IgA compared to C3H/HeJ, which was accompanied by stark differences in gut microbiome composition. CC027/GeniUnc mice mounted antigen-specific IgE responses to peanut, walnut and egg, but not milk, while C3H/HeJ mice were not sensitized to any antigen. After oral challenge, peanut- and walnut-sensitized CC027/GeniUnc mice experienced anaphylaxis, whereas milk- and egg-sensitized mice did not. Major allergens were detected in serum collected post-challenge from peanut-sensitized mice, but not milk- and egg-sensitized mice. Machine learning on the change in gut microbiome composition after sensitization identified a unique signature in CC027/GeniUnc mice that experienced anaphylaxis, including the depletion of Akkermansia. Overall, these results demonstrate the factors contributing to enteral sensitization in CC027/GeniUnc mice, including diminished fecal IgA, increased allergen absorption and altered gut microbiome composition.

AbstractIntroductionAlpha‐gal syndrome (AGS) is characterized by delayed hypersensitivity to non‐primate mammalian meat in people having specific immunoglobulin E (sIgE) to the oligosaccharide galactose‐alpha‐1,3‐galactose. AGS has been linked to tick bites from Amblyomma americanum (Aa) in the U.S. A small animal model of meat allergy is needed to study the mechanism of alpha‐gal sensitization, the effector phase leading to delayed allergic responses and potential therapeutics to treat AGS.MethodsEight‐ to ten‐weeks old mice with a targeted inactivation of alpha‐1,3‐galactosyltransferase (AGKO) were injected intradermally with 50 μg of Aa tick salivary gland extract (TSGE) on days 0, 7, 21, 28, 42, and 49. Total IgE and alpha‐gal sIgE were quantitated on Day 56 by enzyme‐linked immunosorbent assay. Mice were challenged orally with 400 mg of cooked pork kidney homogenate or pork fat. Reaction severity was assessed by measuring a drop in core body temperature and scoring allergic signs.ResultsCompared to control animals, mice treated with TSGE had 190‐fold higher total IgE on Day 56 (0.60 ± 0.12 ng/ml vs. 113.2 ± 24.77 ng/ml; p < 0.001). Alpha‐gal sIgE was also produced in AGKO mice following TSGE sensitization (undetected vs. 158.4 ± 72.43 pg/ml). Further, sensitized mice displayed moderate clinical allergic signs along with a drop in core body temperature of ≥2°C as an objective measure of a systemic allergic reaction. Interestingly, female mice had higher total IgE responses to TSGE treatment but male mice had larger declines in mean body temperature.ConclusionTSGE‐sensitized AGKO mice generate sIgE to alpha‐gal and demonstrate characteristic allergic responses to pork fat and pork kidney. In keeping with the AGS responses documented in humans, mice reacted more rapidly to organ meat than to high fat pork challenge. This mouse model establishes the central role of tick bites in the development of AGS and provides a small animal model to mechanistically study mammalian meat allergy.

The laboratory mouse is the most widely used animal model for biomedical research, due in part to its well-annotated genome, wealth of genetic resources, and the ability to precisely manipulate its genome. Despite the importance of genetics for mouse research, genetic quality control (QC) is not standardized, in part due to the lack of cost-effective, informative, and robust platforms. Genotyping arrays are standard tools for mouse research and remain an attractive alternative even in the era of high-throughput whole-genome sequencing. Here, we describe the content and performance of a new iteration of the Mouse Universal Genotyping Array (MUGA), MiniMUGA, an array-based genetic QC platform with over 11,000 probes. In addition to robust discrimination between most classical and wild-derived laboratory strains, MiniMUGA was designed to contain features not available in other platforms: (1) chromosomal sex determination, (2) discrimination between substrains from multiple commercial vendors, (3) diagnostic SNPs for popular laboratory strains, (4) detection of constructs used in genetically engineered mice, and (5) an easy-to-interpret report summarizing these results. In-depth annotation of all probes should facilitate custom analyses by individual researchers. To determine the performance of MiniMUGA, we genotyped 6899 samples from a wide variety of genetic backgrounds. The performance of MiniMUGA compares favorably with three previous iterations of the MUGA family of arrays, both in discrimination capabilities and robustness. We have generated publicly available consensus genotypes for 241 inbred strains including classical, wild-derived, and recombinant inbred lines. Here, we also report the detection of a substantial number of XO and XXY individuals across a variety of sample types, new markers that expand the utility of reduced complexity crosses to genetic backgrounds other than C57BL/6, and the robust detection of 17 genetic constructs. We provide preliminary evidence that the array can be used to identify both partial sex chromosome duplication and mosaicism, and that diagnostic SNPs can be used to determine how long inbred mice have been bred independently from the relevant main stock. We conclude that MiniMUGA is a valuable platform for genetic QC, and an important new tool to increase the rigor and reproducibility of mouse research.

The incidence and prevalence of allergic reactions to foods are increasing, although the reasons are not understood. The natural history of food allergy varies, as some resolve spontaneously while others persist throughout life. This chapter reviews the major allergens of important food sources that are identified and well characterized at the molecular level. The chapter also discusses taxonomy and classification of animal and plant food allergens based on their protein families. Diagnostic methods for IgE-induced food allergens currently available and novel methods still under investigation are discussed. The current state of food allergy therapeutics—early introduction of allergenic foods, oral immunotherapy, sublingual immunotherapy, epicutaneous immunotherapy, and anti-IgE therapies—are reviewed along with a glimpse of emerging therapies currently in preclinical phases. Although several experimental treatments are promising, none are currently available for the practicing physician.

Purpose of Review Food allergy is a growing health problem worldwide that impacts millions of individuals. Current treatment options are limited and strict dietary avoidance remains the standard of care. Immunotherapy using whole, native allergens is under active clinical investigation but harbors the risk of severe side effects including anaphylaxis. Newer food-specific therapies with hypoallergenic proteins may potentially offer safer treatment alternatives, and this review seeks to investigate the evidence supporting the use of these modalities. Recent Findings The utilization of different methods to alter allergen structure and IgE binding leads to reduced allergenicity and decreases the risk for systemic reactions, making the use of potential therapies including extensively heated egg/milk, peptide immunotherapy, recombinant allergen immunotherapy, and DNA vaccines safe and possibly efficacious forms of treatment in food allergy. However, for the majority of these treatment modalities, limited data currently exists looking at the safety and efficacy in human subjects with food allergy. Summary This review provides a comprehensive overview of the current evidence examining the safety and efficacy of hypoallergenic proteins in the treatment of food allergies.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a naïve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Recently, we identified a Collaborative Cross mouse strain (CC027) that is orally reactive to peanut following sensitization with peanut plus cholera toxin. Interestingly, these mice were found to produce detectable levels of peanut-specific IgE before sensitization with peanut and cholera toxin. Accordingly, we aimed to determine whether CC027 mice could be sensitized to peanut without the use of any adjuvant. CC027 female mice aged 4-6 weeks were sensitized intragastrically with peanut extract once per week or three times per week for four weeks. Mice were bled for IgE measurements and orally challenged with peanut. Body temperature decreases during the peanut challenge demonstrated that mice sensitized without an adjuvant reacted to peanut, and the frequency per week determined reaction severity. Mice sensitized with peanut three times per week had significantly larger decreases in temperature (mean -4.5 °C) compared to mice sensitized only once per week (mean -1.6 °C) (p=0.0065). Peanut-specific IgE levels increased in mice treated three times per week compared to naïve mice (p=0.0095). CC027 mice can be sensitized through the gastrointestinal tract with peanut in the absence of strong mucosal adjuvants. Mice given peanut three times per week were more allergic than mice sensitized only once per week indicating that frequency of antigen administration may drive severity of allergy. Previous reports have demonstrated that mice can be sensitized to peanut epicutaneously, whereas this model is the first to show that mice can be sensitized intragastrically to peanut without an adjuvant, which may mimic peanut sensitization in humans.

Oral, sublingual, and epicutaneous immunotherapies are under clinical study as potential food allergy therapies, yet the side effects, requirement for daily dosing, and lack of prolonged efficacy remain limitations in these human trials.1Jones S.M. Burks A.W. Dupont C. State of the art on food allergen immunotherapy: oral, sublingual, and epicutaneous.J Allergy Clin Immunol. 2014; 133: 318-323Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 2Gorelik M. Narisety S.D. Guerrerio A.L. Chichester K.L. Keet C.A. Bieneman A.P. et al.Suppression of the immunologic response to peanut during immunotherapy is often transient.J Allergy Clin Immunol. 2015; 135: 1283-1292Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Targeting the allergen-specific B cells may limit side effects and promote long-term tolerance. Sialic acid–binding immunoglobulin-type lectins (Siglecs) are a family of immunomodulatory receptors with cell-specific expression.3Macauley M.S. Crocker P.R. Paulson J.C. Siglec-mediated regulation of immune cell function in disease.Nat Rev Immunol. 2014; 14: 653-666Crossref PubMed Scopus (622) Google Scholar Inhibitory Siglecs, including CD22 expressed on B cells, use immunoreceptor tyrosine-based inhibitory motifs to suppress activatory receptors, such as the B-cell receptor (BCR). Enforcing colocalization of CD22 with the BCR, with liposomes that codisplay an antigen and high-affinity CD22 ligand, not only prevents B-cell activation but also induces apoptosis of the antigen-reactive B cells, resulting in robust immunological tolerance due to depletion of the antigen-specific B cells from the B-cell repertoire.4Macauley M.S. Pfrengle F. Rademacher C. Nycholat C.M. Gale A.J. von Drygalski A. et al.Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis.J Clin Invest. 2013; 123: 3074-3083Crossref PubMed Scopus (150) Google Scholar These Siglec-engaging tolerance-inducing antigenic liposomes (STALs) can be formulated with any antigen of choice. STALs displaying factor VIII (FVIII) inhibit antibody responses to exogenous FVIII, preventing bleeding in FVIII−/− mice.4Macauley M.S. Pfrengle F. Rademacher C. Nycholat C.M. Gale A.J. von Drygalski A. et al.Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis.J Clin Invest. 2013; 123: 3074-3083Crossref PubMed Scopus (150) Google Scholar Accordingly, STALs have the potential to prevent undesired B-cell responses and we were motivated to examine their potential for inducing immunological tolerance to a food allergen. Peanut allergies are dominated by undesired IgE antibody responses to the 2S albumin Ara h 2 (Ah2),5Klemans R.J. van Os-Medendorp H. Blankestijn M. Bruijnzeel-Koomen C.A. Knol E.F. Knulst A.C. Diagnostic accuracy of specific IgE to components in diagnosing peanut allergy: a systematic review.Clin Exp Allergy. 2015; 45: 720-730Crossref PubMed Scopus (95) Google Scholar, 6Kulis M. Chen X. Lew J. Wang Q. Patel O.P. Zhuang Y. et al.The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice.Clin Exp Allergy. 2012; 42: 326-336Crossref PubMed Scopus (64) Google Scholar which induce degranulation of effector cells on exposure to the antigen. We hypothesized that STALs displaying both a high-affinity and selective CD22 ligand and Ah2 (Fig 1, A) could be an attractive strategy to prevent sensitization and subsequent anaphylaxis to Ah2 and potentially whole peanut extract (WPE). A schematic representing the experimental design is shown in Fig 1, B. All animal studies were approved by the University of North Carolina Institutional Animal Care and Use Committee and investigated under protocol number 13-216.0. Four-week-old female BALB/cJ mice (Jackson Laboratories, Bar Harbor, Me) were injected intravenously with 200 μL of 100 μM Ah2 STALs (n = 8), 300 μM Ah2 STALs (n = 8), 100 μM immunogenic Ah2 liposomes (n = 8), or 300 μM immunogenic Ah2 liposomes (n = 7). All liposomes consisted of 0.03 mol% Ah2, which amounted to a dose of 0.12 μg of Ah2 in the 100-μM group. STALs also consisted of 1% BPA-Neu5Gc, the high-affinity and selective CD22 ligand.4Macauley M.S. Pfrengle F. Rademacher C. Nycholat C.M. Gale A.J. von Drygalski A. et al.Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis.J Clin Invest. 2013; 123: 3074-3083Crossref PubMed Scopus (150) Google Scholar Two weeks following infusion of STALs, a time frame previously determined to maximize tolerance induction through STALs,4Macauley M.S. Pfrengle F. Rademacher C. Nycholat C.M. Gale A.J. von Drygalski A. et al.Antigenic liposomes displaying CD22 ligands induce antigen-specific B cell apoptosis.J Clin Invest. 2013; 123: 3074-3083Crossref PubMed Scopus (150) Google Scholar the mice were orally sensitized, with 2 mg WPE and 10 μg cholera toxin (CT) weekly for 3 weeks followed by a boost dose of 5 mg WPE and 10 μg CT. A group of naive mice (n = 8) underwent the same protocol and were injected with PBS to determine baseline titers. Serum was collected 1 week later to quantify specific IgE (sIgE) and sIgG1 levels to Ah2, WPE, Ah1, and CT by ELISA.6Kulis M. Chen X. Lew J. Wang Q. Patel O.P. Zhuang Y. et al.The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice.Clin Exp Allergy. 2012; 42: 326-336Crossref PubMed Scopus (64) Google Scholar Mice were initially challenged with 200 μg Ah2 via an intraperitoneal injection. One week later, mice were challenged intraperitoneally with 750 μg WPE. To assess anaphylaxis during challenge, rectal temperatures were recorded for 30 minutes, and symptom scores were documented at 30 minutes using a 0- to 5-point scale, where 0 represents no symptoms and 5 represents death, as described previously.6Kulis M. Chen X. Lew J. Wang Q. Patel O.P. Zhuang Y. et al.The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice.Clin Exp Allergy. 2012; 42: 326-336Crossref PubMed Scopus (64) Google Scholar All methods are described in detail in this article's Online Repository at www.jacionline.org. On day 42, before the challenge, Ah2 sIgE levels were significantly lower in animals injected with either 100 μM or 300 μM Ah2 STALs compared with those injected with the same dose of immunogenic controls (100 μM, P = .0002; 300 μM, P = .0006) (Fig 1, C). Pretreatment with Ah2 STALs also inhibited production of Ah2 sIgG1 compared with controls (100 μM, P = .0047; 300 μM, P = .0006) (Fig 1, D). Upon challenge with 200 μg Ah2, mice pretreated with either 100 μM or 300 μM Ah2 STALs were protected from hypothermia, an objective feature of anaphylaxis in mice, compared with mice pretreated with immunogenic control that had severe reactions (Fig 1, E). The symptom scores, as defined previously,6Kulis M. Chen X. Lew J. Wang Q. Patel O.P. Zhuang Y. et al.The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut-allergic mice.Clin Exp Allergy. 2012; 42: 326-336Crossref PubMed Scopus (64) Google Scholar also reflected more severe reactions in animals pretreated with immunogenic Ah2 liposomes compared with those pretreated with Ah2 STALs (Fig 1, F; 100 μM, P = .0126; 300 μM, P = .0002). CT sIgE was not different among treatment groups (Fig E1), indicating that tolerance induction is antigen-specific and that no intrinsic differences in an ability to mount antibody responses were present between the groups. These findings validated the results of a pilot study where 100 μM but not 20 μM Ah2 STALs led to significantly lower Ah2-specific IgE and IgG1 levels. Taken together, these results suggest that Ah2 STALs induce antigen-specific tolerance toward the major peanut allergen, severely blunting Ah2 sIgE and sIgG1 levels and reactions upon intraperitoneal challenge with Ah2. Because Ah2 is only 1 of several related antigens in peanuts and sensitization was done with WPE, we also examined the impact of Ah2 STALs on sIgE levels to WPE (Fig 2, A) and to another peanut allergen, Ara h 1 (Ah1). Pretreatment with 100 μM Ah2 STALs by intravenous injection resulted in lower peanut sIgE level compared with the immunogenic control mice, though differences were not statistically significant (P = .1296). However, mice that received 300 μM Ah2 STALs (intravenous) had significantly less peanut sIgE than did their control counterparts (P = .0037). Interestingly, mice injected with Ah2 STALs had lower Ah1 sIgE levels than did their controls at each dose (100 μM, P = .0146; 300 μM, P = .0140; Fig 2, B). Extensive cross-reactivity between Ah1 and Ah2 has previously been demonstrated and is likely to account for this effect.7Bublin M. Kostadinova M. Radauer C. Hafner C. Szepfalusi Z. Varga E.M. et al.IgE cross-reactivity between the major peanut allergen Ara h 2 and the nonhomologous allergens Ara h 1 and Ara h 3.J Allergy Clin Immunol. 2013; 132: 118-124Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 8Smit J.J. Pennings M.T. Willemsen K. van Roest M. van Hoffen E. Pieters R.H. Heterogeneous responses and cross reactivity between the major peanut allergens Ara h 1, 2, 3 and 6 in a mouse model for peanut allergy.Clin Transl Allergy. 2015; 5: 13Crossref PubMed Scopus (11) Google Scholar Finally, these mice were challenged with 750 μg WPE and, similar to the WPE sIgE results, the body temperatures of mice pretreated with 300 μM Ah2 STALs were significantly greater than those of mice that received 300 μM immunogenic controls, demonstrating that the STALs attenuated anaphylaxis to WPE (Fig 2, C). Symptom scores reflected similar results, with the 300 μM immunogenic control mice reacting more severely than the Ah2 STALs mice (P = .0350). The groups treated with 100 μM Ah2 STALs were not significantly different in body temperatures or symptom scores from the immunogenic control group, suggesting a dose effect. These results demonstrate for the first time that antigen-specific B cells for a single component can be selectively targeted to diminish an immune response to a complex mixture of several allergens. In conclusion, our data demonstrate that liposomes simultaneously targeting CD22 and the BCR specific for the major peanut allergen, Ah2, can be used to prevent sensitization to Ah2. On the basis of previous studies using other antigens, we hypothesize that simultaneous engagement of CD22 and the Ah2-specific BCR leads to deletion of the Ah2 B cells. Future experiments include testing a combination of STALs that display several peanut antigens as well as using Ah2 STALs as a postsensitization therapy. These findings provide the foundation for the development of a novel therapy for peanut allergy using a highly targeted, antigen-specific approach. Peanut proteins were extracted by mixing peanut flour (12% fat light roast, 50% protein; Golden Peanut Co, Alpharetta, Ga) in a 1:5 (wt:vol) ratio of PBS with 1 mol/L NaCl. The solution was mixed for 2 hours while maintaining an alkaline pH (8.5). The solution was centrifuged at 14,000 rpm for 45 minutes at 4°C. The supernatant was collected and filter-sterilized through a 0.2-μM filter. Protein concentration was determined by bicinchoninic acid assay (Pierce, Rockford, Ill). The final preparation is referred to in the article as WPE. Ah2 was purified according to work previously published by Sen et al.E1Sen M. Kopper R. Pons L. Abraham E.C. Burks A.W. Bannon G.A. Protein structure plays a critical role in peanut allergen stability and may determine immunodominant IgE-binding epitopes.J Immunol. 2002; 169: 882-887Crossref PubMed Scopus (205) Google Scholar A frozen stock of Ah2 in dH2O was initially buffer exchanged into PBS. The protein (2 mg/mL) was reacted with 2.5 molar equivalents of succinimidyl 3-(2-pyridyldithio) propionate (Thermo Fisher, Waltham, Mass) for 1 hour at room temperature, followed by desalting over Sephadex G-50 (GE Healthcare Life Sciences, Malborough, Mass). Following deprotection with 25 mM dithiothreitol for 10 minutes at room temperature, the protein was again desalted. The thiol-modified protein was reacted with 10 equivalents of maleimide-PEG2000-distearoylphosphatidylethanolamine (DSPE; NOF America, Irvine, Calif) overnight under nitrogen. The following day, the reaction was passed over a Sephadex G-100 column (GE Healthcare) and the fractions containing the lipid-modified Ah2 in the void volume were pooled and stored at 4°C. Distearoylphosphatidylcholine (Avanti, Alabaster, Ala), cholesterol (Sigma-Aldrich, St Louis, Mo), and PEG2000-DSPE (NOF America) were dissolved in chloroform and combined at an approximate ratio of 57:38:5. The solvent was evaporated under nitrogen and 100 μL of dimethyl sulfoxide (DMSO) was added. To formulate the STALs, 1 mol% of PEG2000-DSPE was replaced with the high-affinity CD22 ligand (6′BPA-Neu5Gc-LacNAc-PEG2000-DSPE) and added from a DMSO stock. The lipid solutions in DMSO were lyophilized overnight to yield a fluffy powder. To hydrate the liposomes, the dried lipids were hydrated in the appropriate amount of PBS containing the Ah2-PEG-DSPE such that the mol% of the protein was 0.033 mol% of the total lipids. The lipids were hydrated in 1 mL to generate liposomes with a concentration of 5 mM total lipid. The hydrated lipids were sonicated a minimum of 5 times at 30-second intervals and then extruded using a hand-extruder (Avanti) 20 times through a 0.8-μM filter, followed by 20 times through a 0.1-μM filter. The final liposomes passed over a CL-4B column (Sigma) and pooled fractions were stored at 4°C before being diluted in PBS to the appropriate liposome concentration and administered to the mice. Four-week-old female Balb/cJ mice purchased from Jackson Laboratory were maintained on peanut-free food under pathogen-free conditions. Mice were housed with 4 animals per cage and separated into 5 groups: (1) naive, (2) 100 μM Ah2 immunogenic liposomes, (3) 100 μM Ah2 STALs, (4) 300 μM immunogenic liposomes, and (5) 300 μM Ah2 STALs. Naive mice were maintained throughout the experiment but received no treatments and were not sensitized. Mice in groups 2 to 5 received a single tail vein injection of the designated liposome or STAL. Mice were rested for 2 weeks before being sensitized. After the 2-week rest period, mice were sensitized with 2 mg peanut extract and 10 μg CT (List Biological Laboratories, Campbell, Calif) by oral gavage once a week for 3 weeks. On the fourth week of sensitization, mice were boosted with 1 gavage of 5 mg peanut extract and 10 μg CT. One week after the last sensitization dose, 200 μL blood from each mouse was collected by submandibular bleed to measure sIgE and IgG1 levels by ELISA. The day following bleeding, mice in groups 2 to 5 were challenged with 200 μg intraperitoneal Ah2 and 1 week later were challenged intraperitoneally with 750 μg peanut extract. Anaphylactic reactions were assessed by measuring core body temperatures with a rectal probe (Physitemp, Clifton, NJ) at 15-minute intervals. Symptoms were scored after 30 minutes using a pre-established 5-point scale: 0, no symptoms; 1, scratching around the nose and head; 2, puffiness around the eyes and mouth with reduced activity; 3, labored respiration and/or cyanosis around the mouth and tail; 4, no activity after prodding or tremor and convulsion; and 5, death. sIgE and IgG1 levels were measured by ELISA using a reference cure. Plates were coated with 20 μg/mL WPE, 5 μg/mL Ah2, 5 μg/mL Ah1, or 5 μg/mL CT in carbonate-bicarbonate buffer at pH 9.6 for 1 hour at 37°C. Plates were blocked with PBS containing 0.05% Tween 20 and 2% BSA for 2 hours at 37°C. Serum samples were added for 1 hour at 37°C. Detection of IgE was performed using sheep antimouse IgE (0.5 μg/mL; Binding Site, Birmingham, United Kingdom), followed by biotinylated donkey antisheep IgG (0.5 μg/mL; Accurate Chemical, Westbury, NY) and neutravidin-horseradish peroxidase (HRP; 0.2 μg/mL; Pierce) for 1 hour at 37°C. IgG1 was detected by HRP-conjugated goat anti-mouse IgG1 (Southern Biotech, Birmingham, Ala) used at 1:40,000 for 1 hour at 37°C. HRP activity was measured by blue color development of Sure Blue TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, Md). Plates were read on an Epoch Microplate Spectrophotometer (BioTek Instruments, Winooski, Vt).

B-cell receptors (BCRs) play a critical role in adaptive immunity as they generate highly diverse immunoglobulin repertoires to recognize a wide variety of antigens. To better understand immune responses, it is critically important to establish a quantitative and rapid method to analyze BCR repertoire comprehensively. Here, we developed "Bcrip", a novel approach to characterize BCR repertoire by sequencing millions of BCR cDNA using next-generation sequencer. Using this method and quantitative real-time PCR, we analyzed expression levels and repertoires of BCRs in a total of 17 peanut allergic subjects' peripheral blood samples before and after receiving oral immunotherapy (OIT) or placebo. By our methods, we successfully identified all of variable (V), joining (J), and constant (C) regions, in an average of 79.1% of total reads and 99.6% of these VJC-mapped reads contained the C region corresponding to the isotypes that we aimed to analyze. In the 17 peanut allergic subjects' peripheral blood samples, we observed an oligoclonal enrichment of certain immunoglobulin heavy chain alpha (IGHA) and IGH gamma (IGHG) clones (P = 0.034 and P = 0.027, respectively) in peanut allergic subjects after OIT. This newly developed BCR sequencing and analysis method can be applied to investigate B-cell repertoires in various research areas, including food allergies as well as autoimmune and infectious diseases.

Oral immunotherapy (OIT) can promote desensitization to food allergens, but minimizing, or lessening in severity, the high rate of adverse reactions is a clinical imperative.1 The repetitive oral administration of allergens to atopic individuals mimics exposures thought to trigger eosinophilic esophagitis (EoE). Indeed, several cases of EoE occurring during OIT have been noted,2,3 including during peanut OIT with omalizumab, a strategy intended to reduce side effects and to allow for faster desensitization.