Michael D. Kulis

Sustained unresponsiveness to peanut in subjects who have completed peanut oral immunotherapy

BACKGROUND Although peanut oral immunotherapy (OIT) has been conclusively shown to cause desensitization, it is currently unknown whether clinical protection persists after stopping therapy. OBJECTIVE Our primary objective was to determine whether peanut OIT can induce sustained unresponsiveness after withdrawal of OIT. METHODS We conducted a pilot clinical trial of peanut OIT at 2 US centers. Subjects age 1 to 16 years were recruited and treated for up to 5 years with peanut OIT. The protocol was modified over time to permit dose increases to a maximum of 4000 mg/d peanut protein. Blood was collected at multiple time points. Clinical end points were measured with 5000-mg double-blinded, placebo-controlled food challenges once specific criteria were met. RESULTS Of the 39 subjects originally enrolled, 24 completed the protocol and had evaluable outcomes. Twelve (50%) of 24 successfully passed a challenge 1 month after stopping OIT and achieved sustained unresponsiveness. Peanut was added to the diet. At baseline and the time of challenge, such subjects had smaller skin test results, as well as lower IgE levels specific for peanut, Ara h 1, and Ara h 2 and lower ratios of peanut-specific IgE/total IgE compared with subjects not passing. There were no differences in peanut IgG₄ levels or functional activity at the end of the study. CONCLUSIONS This is the first demonstration of sustained unresponsiveness after peanut OIT, occurring in half of subjects treated for up to 5 years. OIT favorably modified the peanut-specific immune response in all subjects completing the protocol. Smaller skin test results and lower allergen-specific IgE levels were predictive of successful outcome.

Individualized IgE-based dosing of egg oral immunotherapy and the development of tolerance

BACKGROUND Hens egg allergy is among the most common food allergies in childhood and predicts later development of allergic disease. The optimal efficacy and mechanism(s) of egg allergen immunotherapy are poorly understood. OBJECTIVE To enhance immunologic and clinical outcomes of egg oral immunotherapy (OIT) using a conditionally increased dosing strategy. METHODS In an open-label clinical trial of egg OIT, egg-allergic children ingested daily doses of egg protein that were gradually increased based on the egg white (EW) IgE level. Skin prick test reactivity and EW- and ovomucoid-specific cellular and humoral responses were measured longitudinally. To confirm clinical tolerance, patients underwent double-blinded, placebo-controlled food challenges 1 month after completing the dosing protocol. RESULTS Children aged 3 to 13 years with characteristics of clinical egg allergy were enrolled. All 6 patients who completed the entire protocol developed clinical tolerance to egg during the study. The median wheal diameter on EW skin prick testing decreased from 10 to 2.5 mm during OIT (P = .03). Both EW and ovomucoid IgE levels significantly decreased during the study (median EW IgE level: from 18.8 kU/L at baseline to 3.9 kU/L, P = .03), and corresponding IgG4 levels increased (median EW IgG4 level: from 0.65 mg/L at baseline to 86.15 mg/L, P = .03). Transient increases were seen in egg-induced interleukin 10 (P = .06) and transforming growth factor β (P = .18) levels, and the ratio of T(H)2:T(H)1 cytokine production was decreased (P = .25). CONCLUSIONS Egg OIT is associated with tolerance acquisition in children with persistent egg allergy. Individualized dosing regimens may be necessary to achieve a full therapeutic effect in some patients.

Peanut oral immunotherapy modifies IgE and IgG4 responses to major peanut allergens.

BACKGROUND Patients with peanut allergy have highly stable pathologic antibody repertoires to the immunodominant B-cell epitopes of the major peanut allergens Ara h 1 to 3. OBJECTIVE We used a peptide microarray technique to analyze the effect of treatment with peanut oral immunotherapy (OIT) on such repertoires. METHODS Measurements of total peanut-specific IgE (psIgE) and peanut-specific IgG(4) (psIgG(4)) were made with CAP-FEIA. We analyzed sera from 22 patients with OIT and 6 control subjects and measured serum specific IgE and IgG(4) binding to epitopes of Ara h 1 to 3 using a high-throughput peptide microarray technique. Antibody affinity was measured by using a competitive peptide microarray, as previously described. RESULTS At baseline, psIgE and psIgG(4) diversity was similar between patients and control subjects, and there was broad variation in epitope recognition. After a median of 41 months of OIT, polyclonal psIgG(4) levels increased from a median of 0.3 μg/mL (interquartile range [25% to 75%], 0.1-0.43 μg/mL) at baseline to 10.5 μg/mL (interquartile range [25% to 75%], 3.95-45.48 μg/mL; P < .0001) and included de novo specificities. psIgE levels were reduced from a median baseline of 85.45 kU(A)/L (23.05-101.0 kU(A)/L) to 7.75 kU(A)/L (2.58-30.55 kU(A)/L, P < .0001). Affinity was unaffected. Although the psIgE repertoire contracted in most OIT-treated patients, several subjects generated new IgE specificities, even as the total psIgE level decreased. Global epitope-specific shifts from IgE to IgG(4) binding occurred, including at an informative epitope of Ara h 2. CONCLUSION OIT differentially alters Ara h 1 to 3 binding patterns. These changes are variable between patients, are not observed in control subjects, and include a progressive polyclonal increase in IgG(4) levels, with concurrent reduction in IgE amount and diversity.

Sublingual versus oral immunotherapy for peanut-allergic children: A retrospective comparison

To the Editor: There has been considerable recent interest in developing therapies for food allergy, an increasingly common and highly morbid disorder for which strict dietary elimination and ready access to epinephrine remain the standard of care.(1) While both oral immunotherapy (OIT)(2-4) and sublingual immunotherapy (SLIT)(5) have been shown to induce clinical desensitization to foods (reviewed in 6), no head-to-head comparative analysis of the two treatments has been published. We conducted a retrospective study of two previously published protocols for peanut allergy(2,3). This new analysis includes additional subjects, compares the 12-month oral food challenge outcomes, and extends analysis of immunologic parameters out to 24 months. Eligible peanut-allergic subjects were recruited into one of two concurrent clinical trials: OIT (maintenance dose of 4000 mg/day and cumulative double-blind, placebo-controlled challenge (DBPCFC) dose of 5000 mg); or SLIT, (2 mg/day, and 2500 mg, respectively) [all quantities refer to peanut protein]. Although the optimal immunotherapy dose remains unknown, the doses chosen in these trials were based on preliminary data from pilot studies. Of note, unique properties of the oral mucosal immune response are hypothesized to account for SLIT’s efficacy at log-fold lower doses (reviewed in 7). Both trials utilized randomized, double-blind, placebo-controlled designs. Mechanistic studies were performed longitudinally as previously described using blood drawn from subjects within 24 hours of their last immunotherapy dose.(2, 3) At 12 months, subjects underwent DBPCFC to assess clinical desensitization; OIT subjects received a maximal 5000 mg cumulative protein dose, and for safety reasons SLIT challenges were limited to 2500 mg (Online Repository Tables E1/E2). We compared laboratory data between OIT and SLIT at baseline, 12 months, and 24 months, as well as DBPCFC pass/fail outcomes, using the Wilcoxon signed rank test (STATA 12; College Station, TX) and Mann-Whitney U test (GraphPad Prism; La Jolla, CA). Twenty-three subjects on OIT and 27 subjects on SLIT were evaluated after receiving 2 years of treatment (Table 1). We did not undertake a formal comparison of safety parameters between the two studies, and upcoming interval reports of each study will include these data. However, there were no serious adverse events reported in either study. No SLIT and two OIT subjects (one active, one placebo) required four total doses of epinephrine for dose-related reactions. At baseline, the peanut-specific IgE was similar between OIT and SLIT subjects (Fig 1A). Twelve months of treatment led to higher median peanut-specific IgE levels in the OIT group compared to the SLIT group (204.5 kU/L versus 66.7 kU/L, p=0.0382); however, levels were not significantly different between the groups at 24 months (Fig 1A). While peanut-specific IgG4 increased over time in both groups (Fig 1B), the effect was greater with OIT at 12 (20.1 mg/L versus 3.1 mg/L) and 24 months (20.3 mg/L versus 7.9 mg/L, p<0.001). Although decreased in both groups, median peanut-specific IgE/IgG4 ratios were significantly lower at 12 and 24 months for subjects receiving OIT (Fig 1C). Thirty-four subjects (14 OIT, 20 SLIT) had basophil activation assessed by CD63 up-regulation at baseline and 12 months. After 12 months, a significantly lower percentage of CD63+ basophils was found in the OIT group compared with the SLIT group when stimulated with 100 (median 5.90% versus 21.50%) and 10−1 μg/mL (median 6.34% versus 30.75%) crude peanut extract (p<0.01). No between-group difference was seen after stimulation with weaker dilutions of 10−2 and 10−3 μg/mL crude peanut extract. Too few samples were obtained at 24 months to perform an analysis. FIG 1 A and B, Change in serum peanut-IgE and peanut-IgG4 (SLIT/OIT). C, IgE/IgG4 ratio to peanut (SLIT/OIT). D, Cumulative amount tolerated during DBPCFC (SLIT/OIT). E, Serum peanut-IgE (Pass/Fail). F, Fold change in serum peanut-IgG4 from baseline to 12 months ... Table 1 Baseline subject characteristics Eighteen subjects on OIT and 27 subjects on SLIT underwent 12 month desensitization DBPCFCs, results of which are shown in Figure 1D. Despite differences in DBPCFC protocols, SLIT subjects reacted at lower eliciting dose thresholds. A Fisher’s exact test was used to calculate the difference in proportions and relative risk for passing or failing the DBPCFC according to treatment group. The difference in proportions was statistically significant (p=0.002), with OIT-treated subjects 3 times more likely to pass the 12 month desensitization DBPCFC than SLIT-treated subjects (RR=3.00, 95% CI 1.64-5.49). In an attempt to identify candidate biomarkers, we combined all SLIT and OIT subjects and then categorized them by “pass” or “fail” based upon their ability to complete the DBPCFC without symptoms. Consistent with other studies, subjects passing the 12 month desensitization DBPCFC tended to have lower baseline peanut-specific IgE levels (34.6 kU/L versus 167 kU/L, p=0.0575) (Fig 1E). Peanut-specific IgG4 was increased by 27-fold in the “pass” group compared to a 6.5-fold increase in the “fail” group (p=0.01; Fig 1F). Interestingly, the percentage of CD63+ basophils was significantly lower at 12 months in the “pass” group compared with the “fail” group when stimulated with 100 (median 5.90% versus 21.50%) and 10−1 μg/mL (median 6.34% versus 34.75%) crude peanut extract (p<0.01). Again no differences were seen between groups after stimulation with weaker dilutions. Skin prick tests decreased over time in all subjects. Wheal size, serum peanut-specific IgA and peanut-specific IgG, and CD4+CD25+FoxP3+ T-regulatory cells were not significantly different between the OIT and SLIT groups or the “pass” and “fail” groups. In summary, our results suggest that after two years of treatment, OIT produces greater immunologic changes than SLIT in peanut-allergic children. Specifically, peanut OIT resulted in greater changes in peanut-specific IgE, IgG4, and IgE/IgG4 ratio as well as basophil activation. In addition, eliciting dose thresholds were lower and more variable during DBPCFC at 12 months in SLIT-treated subjects, compared to OIT-treated subjects. Subjects who passed the DBPCFC tended to have lower baseline peanut-IgE levels, in addition to a larger fold change in peanut-IgG4 and less basophil activation at 12 months. The major limitation of this study is that it was not a randomized prospective study designed to directly compare the two modalities with a uniform protocol and consecutive enrollment. It is important to also note that interim clinical endpoints measured after only 12 months of immunotherapy likely do not provide a full assessment of the efficacy of either method. Further research is needed to determine the optimal length of treatment, dose, and ideal immunotherapy candidate for each modality.

Early oral immunotherapy in peanut-allergic preschool children is safe and highly effective

Background: Oral immunotherapy (OIT) is an effective experimental food allergy treatment that is limited by treatment withdrawal and the frequent reversibility of desensitization if interrupted. Newly diagnosed preschool children may have clinical and immunological characteristics more amenable to treatment. Objective: We sought to test the safety, effectiveness, and feasibility of early OIT (E‐OIT) in the treatment of peanut allergy. Methods: We enrolled 40 children aged 9 to 36 months with suspected or known peanut allergy. Qualifying subjects reacted to peanut during an entry food challenge and were block‐randomized 1:1 to receive E‐OIT at goal maintenance doses of 300 or 3000 mg/d in a double‐blinded fashion. The primary end point, sustained unresponsiveness at 4 weeks after stopping early intervention oral immunotherapy (4‐SU), was assessed by double‐blinded, placebo‐controlled food challenge either upon achieving 4 prespecified criteria, or after 3 maintenance years. Peanut‐specific immune responses were serially analyzed. Outcomes were compared with 154 matched standard‐care controls. Results: Of 40 consented subjects, 3 (7.5%) did not qualify. Overall, 29 of 37 (78%) in the intent‐to‐treat analysis achieved 4‐SU (300‐mg arm, 17 of 20 [85%]; 3000 mg, 12 of 17 [71%], P = .43) over a median of 29 months. Per‐protocol, the overall proportion achieving 4‐SU was 29 of 32 (91%). Peanut‐specific IgE levels significantly declined in E‐OIT‐treated children, who were 19 times more likely to successfully consume dietary peanut than matched standard‐care controls, in whom peanut‐specific IgE levels significantly increased (relative risk, 19.42; 95% CI, 8.7‐43.7; P < .001). Allergic side effects during E‐OIT were common but all were mild to moderate. Conclusions: At both doses tested, E‐OIT had an acceptable safety profile and was highly successful in rapidly suppressing allergic immune responses and achieving safe dietary reintroduction.

Increased peanut-specific IgA levels in saliva correlate with food challenge outcomes after peanut sublingual immunotherapy

Capsule Summary Peanut-specific IgA in saliva correlates with DBPCFC outcomes following peanut SLIT, suggesting that peanut-specific salivary IgA may be a potential biomarker for SLIT used to treat peanut allergy.

Immunomodulatory Gene Therapy Prevents Antibody Formation and Lethal Hypersensitivity Reactions in Murine Pompe Disease

Infantile Pompe disease progresses to a lethal cardiomyopathy in absence of effective treatment. Enzyme-replacement therapy (ERT) with recombinant human acid alpha-glucosidase (rhGAA) has been effective in most patients with Pompe disease, but efficacy was reduced by high-titer antibody responses. Immunomodulatory gene therapy with a low dose adeno-associated virus (AAV) vector (2 x 10(10) particles) containing a liver-specific regulatory cassette significantly lowered immunoglobin G (IgG), IgG1, and IgE antibodies to GAA in Pompe disease mice, when compared with mock-treated mice (P < 0.05). AAV-LSPhGAApA had the same effect on GAA-antibody production whether it was given prior to, following, or simultaneously with the initial GAA injection. Mice given AAV-LSPhGAApA had significantly less decrease in body temperature (P < 0.001) and lower anaphylactic scores (P < 0.01) following the GAA challenge. Mouse mast cell protease-1 (MMCP-1) followed the pattern associated with hypersensitivity reactions (P < 0.05). Regulatory T cells (Treg) were demonstrated to play a role in the tolerance induced by gene therapy as depletion of Treg led to an increase in GAA-specific IgG (P < 0.001). Treg depleted mice were challenged with GAA and had significantly stronger allergic reactions than mice given gene therapy without subsequent Treg depletion (temperature: P < 0.01; symptoms: P < 0.05). Ubiquitous GAA expression failed to prevent antibody formation. Thus, immunomodulatory gene therapy could provide adjunctive therapy in lysosomal storage disorders treated by enzyme replacement.

Letter to the editorIncreased peanut-specific IgA levels in saliva correlate with food challenge outcomes after peanut sublingual immunotherapy

Capsule Summary Peanut-specific IgA in saliva correlates with DBPCFC outcomes following peanut SLIT, suggesting that peanut-specific salivary IgA may be a potential biomarker for SLIT used to treat peanut allergy.

In vivo and T Cell Cross-Reactivity between Walnut, Cashew and Peanut

Background: Examination of IgE cross-reactivity among nuts has been limited to in vitro experiments. Cross-reactivity studies of nuts at the T cell level are difficult to interpret because of the inability to determine which cellular responses are from a true sensitization and which are due to cross-reactivity. Using a mouse model in which the sensitizing nuts are controlled may provide novel methods to investigate in vivo and T cell cross-reactivity. Methods: C3H/HeJ mice were sensitized by intraperitoneal injection of cashew alone (monosensitized mice), or cashew plus walnut, utilizing alum as an adjuvant. Both groups underwent challenges to cashew, walnut and peanut, with subsequent monitoring of anaphylactic reactions. Anaphylactic antibodies were quantified by ELISA, and protein allergens were identified by Western blotting. Cellular responses were studied via splenocyte proliferation assay and measurement of secreted cytokines. Results: The monosensitized mice reacted to cashew and walnut during challenges, with significantly weaker reactions induced on challenge with peanut. Cross-reactive IgE to walnut and peanut were detected by ELISA, and the cross-reactive allergens were identified as vicilin proteins. In cellular assays, splenocytes from the monosensitized mice proliferated and produced IL-4 and IL-5 in response to cashew, walnut and peanut. The cashew- plus walnut-sensitized mice experienced stronger clinical reactions to walnut, recognized additional walnut allergens and secreted significantly more IL-4 and IL-5 in walnut-stimulated splenocyte assays compared to the monosensitized mice. Conclusions: Cross-reactivity in vivo was found between cashew and walnut, while cross-reactivity among cashew, walnut and peanut was demonstrated at the T cell level.

Component-resolved analysis of IgA, IgE, and IgG4 during egg OIT identifies markers associated with sustained unresponsiveness

In a previously reported CoFAR study, 55 subjects with egg allergy underwent randomized, placebo‐controlled egg oral immunotherapy (eOIT). Active treatment induced desensitization in most and sustained unresponsiveness (SU) in a smaller subset. We hypothesized that component‐resolved analysis of IgE, IgG4, IgA, IgA1, and IgA2 may identify potential biomarkers of SU in OIT subjects.