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. Author manuscript; available in PMC: 2025 Sep 1.
Published in final edited form as: J Allergy Clin Immunol. 2024 May 29;154(3):690–697.e4. doi: 10.1016/j.jaci.2024.05.020

Saliva antibody profiles are associated with reaction threshold and severity of peanut allergic reactions

Hsi-en Ho 1, Zoe Arditi 2, Lin Radigan 1, Galina Grishina 3, Lingdi Zhang 2, Yoojin Chun 2, Tracy Lo 3, Julie Wang 3, Scott Sicherer 3, Supinda Bunyavanich 2,3,*
PMCID: PMC11380589  NIHMSID: NIHMS2000746  PMID: 38821318

Abstract

Background:

Reaction threshold and severity in food allergy are difficult to predict, and there is a lack of non-invasive predictors.

Objectives:

We sought to determine the relationships between pre-challenge levels of peanut (PN)-specific antibodies in saliva and reaction threshold, severity, and organ-specific symptoms during peanut allergic reactions.

Methods:

We measured PN-specific antibody levels in saliva collected from 127 children with suspected peanut allergy prior to double-blind, placebo-controlled peanut challenges where reaction threshold, severity, and symptoms were rigorously characterized. Low-threshold peanut allergy was defined as reaction to <300mg of peanut protein cumulatively consumed. A consensus severity grading system was used to grade severity. We analyzed associations between antibody levels and reaction threshold, severity, and organ-specific symptoms.

Results:

Among the 127 children, those with high pre-challenge saliva PN IgE had higher odds of low-threshold peanut allergy (OR 3.9, 95%CI 1.6–9.5), while those with high saliva PN IgA: PN IgE or PN IgG4:PN IgE had lower odds of low-threshold peanut allergy (OR 0.3, 95%CI 0.1–0.8, and OR 0.4, 95%CI 0.2–0.9, respectively). Children with high pre-challenge saliva PN IgG4 had lower odds of severe peanut reactions (OR 0.4, 95%CI 0.2–0.9). Those with high saliva PN IgE had higher odds of respiratory symptoms (OR 8.0, 95%CI 2.2–26.8). Saliva PN IgE modestly correlated with serum PN IgE levels (Pearson r=0.31, P=0.0004). High and low saliva PN IgE levels further distinguished reaction threshold and severity in participants stratified by serum PN IgE, suggesting endotypes.

Conclusion:

Saliva PN antibodies could aid in non-invasive risk stratification of peanut allergy threshold, severity, and organ-specific symptoms.

Keywords: peanut, allergy, food allergy, saliva, non-invasive, reaction, threshold, severity, antibody, symptom, endotype

Capsule summary

Pre-challenge saliva levels of peanut-specific antibodies were associated with reaction threshold, severity, and organ-specific manifestations of peanut allergy, including distinct endotypes. Saliva antibody levels could aid in peanut allergy risk stratification in a non-invasive manner.

Graphical Abstract

graphic file with name nihms-2000746-f0005.jpg

INTRODUCTION

Food allergy is a growing problem globally, with peanut allergy accounting for a large share of food allergy disease burden.1 The quantity of peanut antigen that elicits allergic reactions (i.e. reaction threshold) varies widely between individuals. In addition, the severity of peanut allergy reactions is difficult to predict, ranging from mild symptoms to life-threatening anaphylaxis, with respiratory reactions being the most concerning organ-specific symptom.2 Together, reaction threshold and severity are key parameters of peanut allergy that affect quality of life,3 clinical management (e.g., planning for anaphylaxis),4 as well as risk-stratification for oral food challenges.5 Reaction threshold and severity are also criteria to consider in patient selection for oral immunotherapy in clinical practice.6 Immunologic determinants and predictors of reaction threshold and severity are of great interest for the management and mechanistic understanding of food allergy.5,7

To date, factors involved in varied peanut allergy phenotypes are not fully understood. The search for predictors of peanut allergy reaction threshold and severity has focused primarily on blood analytes.5,7 These include serum peanut (PN) IgE, PN IgG4:IgE ratio, PN component IgE levels, PN epitope-specific IgE, and basophil activation test (BAT).8–12 In a large multi-cohort study that examined all of these blood analytes except for epitope-specific IgE, the best performer for predicting PN reaction threshold and severity was the basophil activation test (BAT).5 However, the need for fresh blood samples and operational barriers have prevented the implementation of BAT in clinical settings.13 Additionally, some patients have non-responder basophils, limiting the universal application of BAT.13

The oral environment is a compelling site to search for immunologic determinants and predictors of peanut allergy reaction threshold and severity. The oral mucosa is the initial interface between ingested food allergens and the immune system.14 As PN allergic reactions can occur within minutes, prior to systemic absorption via the lower gastrointestinal tract, the oral mucosa may be a highly relevant site of action for early and low-threshold reactions. Importantly, saliva sampling is also non-invasive and well-tolerated, even in children, making it a more feasible specimen to collect compared to blood. Saliva antibody levels are candidates to explore as predictors of peanut allergy reaction threshold and severity. While mucosal PN-specific IgA levels have not been linked to protection against future peanut allergy development,15 PN-specific IgA and PN-specific IgG4 in saliva have been shown to increase with desensitization via peanut oral immunotherapy.16 Mechanistically, PN IgG4 and PN IgA may compete with PN IgE for binding to allergens and preventing IgE-mediated mast cell degranulation.17–19

In this study, we examined saliva samples collected from children before double-blind, placebo-controlled peanut challenge to test the hypothesis that pre-challenge saliva antibody profiles might be associated with reaction threshold, severity, and organ-specific symptoms in peanut allergic children.

METHODS

Participant enrollment, sample collection, and double-blind, placebo-controlled peanut challenges

Participants included children undergoing double-blind, placebo-controlled peanut challenges before receiving treatment for two research studies at the Mount Sinai Health System, New York, NY. The same inclusion and exclusion criteria and challenge protocols were used for all participants. Children ages 4–19 years with suspected peanut allergy and evidence of peanut sensitization by skin prick test (SPT) (PN SPT ≥ 3mm larger than saline control) and/or serum PN IgE (PN sIgE ≥ 0.35 kUA/L) were eligible for inclusion. Exclusion criteria included life-threatening anaphylaxis to peanut within the last 2 years, any disorder in which epinephrine is contraindicated, history of chronic disease requiring therapy (other than asthma, atopic dermatitis, rhinitis), on build-up phase of any allergen immunotherapy, uncontrolled asthma, recent systemic steroids, gastrointestinal eosinophilic disorders, therapy with biologics or investigational drugs, pregnancy, and medical problems that in the opinion of the investigator would interfere with participant’s ability to comply with study requirements. The study was approved by the Mount Sinai Institutional Review Board. Participants or the parents of minors provided written informed consent prior to participation, and assent from participating children was also obtained.

Saliva was collected before the double-blind, placebo-controlled challenge. In the 2 hours prior to saliva collection, participants were not permitted to have eaten or drunk liquids, with the exception of water. Unstimulated saliva (0.6 ml) was collected by having the participants hang their head while looking downwards and drip saliva into a collection tube as previously described20; there was no active spitting and no suction was used. Whole saliva samples were placed on ice immediately and stored at −80°C in a 2.0 mL cryotube within 30 minutes of sample collection.

Following saliva collection, participants underwent an allergist-supervised, double-blind, placebo-controlled peanut challenge. Peanut protein doses in the challenge schedule were 3 mg, 10 mg, 30 mg, 100 mg, 300 mg, 600 mg, 1000 mg, 3000 mg, and 4000 mg. Oat flour was used for the placebo challenge. Doses were ingested at 20-minute intervals until reaction or the final dose was given. The order of peanut vs. placebo challenges was randomized, and participants and staff were blinded. Eliciting dose (i.e. the dose ingested that triggered the onset of convincing allergic symptoms) and detailed symptoms were recorded. Challenges were stopped if the criteria for a positive challenge as described in Table E1 were met. Grading of severity was based on a consensus severity grading system for acute allergic reactions as previously described (0, asymptomatic; grade 1–5 from least severe to most severe).21 Participants were classified as having low-threshold peanut allergy if they reacted to less than 300mg of peanut protein cumulatively consumed (Table E2). As reference, the protein content in 1 peanut is 300mg. Children who developed allergy symptoms after cumulatively ingesting more than 300mg of peanut protein were classified as high-threshold.

Participants who did not develop allergy symptoms during the challenge were classified as not peanut allergic. Children with severity grade ≥ 3 were classified as high severity.21

Measurement of saliva PN antibodies

Saliva levels of PN-specific IgE, PN-specific IgA and PN-specific IgG4 were measured via enzyme-linked immunosorbent assays (ELISAs). 96-well plates (Immulon 4HBX microtiter plates, ThermoFisher) were coated with whole peanut protein extract, extracted using a previously described protocol.22 For standard curves, the remaining wells were coated with anti-human IgE (SouthernBiotech, 9160–01), anti-human IgA (SouthernBiotech, 2050–01), or anti-human IgG4 (BD Pharmingen, 555881). Standard curves were generated with native human IgE protein (Abcam, ab152001), native human IgA protein (Abcam, ab91025), or native human IgG4 protein (Abcam, ab183266). Samples and standards were detected with anti-human IgE-HRP (SouthernBiotech, 9160–05), anti-human IgA-HRP (SouthernBiotech, 2050–05), or anti-human IgG4-HRP (SouthernBiotech, 9200–05). Coating buffer, reagent buffer, developing solution, and stopping buffer utilized were from the DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems, DY008). The results were read at 450 nm with FLUOstar Omega multimode microplate reader (BMG Labtech). PN-specific antibody concentrations in units/mL (U/mL) were calculated according to corresponding standard curves. Values below the detection range of the standard curves were valued as 0.5 x lower limit of detection. As check of the assays, we collected saliva from 3 additional children who had food allergies other than peanut allergy and found that they had no detectable saliva PN IgE.

Statistical analyses

As there are limited population data on saliva antibody levels and no established predictive cut-offs for them, we dichotomized saliva antibody levels based on median levels in this cohort. Serum PN IgE levels were dichotomized based on 15 kUA/L, the level associated with 95% certainty of reaction.23 Statistical comparisons were performed using the Fisher’s exact test for categorical variables and Kruskal-Wallis or Mann-Whitney test for continuous variables. Bivariate correlations between continuous variables were calculated using Pearson correlation. All reported P values were 2-sided, and P values less than 0.05 were considered significant. All calculations were performed using GraphPad Prism software (version 8.4.2).

RESULTS

127 children with suspected peanut allergy and median age 8.3 (SD=3.6; range 4–19) years participated in this study, of whom 44% were female (Table 1). All children completed saliva collection before double-blind, placebo-controlled peanut challenge. Twenty seven (21.3%) children developed allergy symptoms after ingesting <300 mg cumulative peanut protein (range 3mg-143mg) and were classified as low-threshold (LT). Seventy eight (61.4%) children developed allergy symptoms after ingesting ≥300 mg of cumulative peanut protein and were classified as high-threshold (HT). The distribution of eliciting peanut dose in the cohort is summarized in Table E2, and symptoms by organ system are detailed in Table E3. Twenty two (17.3%) children did not develop allergy symptoms during the challenge and were classified as not peanut-allergic (NPA). Children with low-threshold peanut allergy had higher peanut-specific serum IgE levels and peanut skin prick test wheal sizes (Table 1). Severity grade ranged from 0–5 (median = 1.7, SD = 1.1). Thirty-three children with severity grade ≥ 3 were classified as high severity. Those with high severity peanut allergy had higher peanut-specific serum IgE levels, but no significant differences in peanut skin prick test wheal sizes (Table 1). There were no significant differences in demographic characteristics, atopic co-morbidities, or other food allergies between the reaction threshold and severity groups. There was no association between severity grade group and reaction threshold group.

Table 1.

Characteristics of the cohort

All (n = 127) Reaction Threshold Severity
NPA (n = 22) High (n = 78) Low (n = 27) P value Low (Grade 0–2) (n = 94) High (Grade 3–5) (n = 33) P value
Age in years, mean (SD) 8.3 (3.6) 8.4 (3.3) 7.7 (3.3) 9.8 (4.3) 0.1 8.3 (3.3) 8.2 (4.2) 0.5
Female sex, no. (%) 56 (44%) 10 (45%) 33 (42%) 13 (48%) 0.86 40 (43%) 16 (48%) 0.55
Race, no. (%) 0.66 0.16
 Asian 22 (17%) 3 (14%) 14 (18%) 5 (19%) 14 (15%) 8 (24%)
 Black 4 (3%) 2 (9%) 1 (1%) 1 (4%) 4 (4%) 0
 Multiple races or other 28 (22%) 4 (18%) 17 (22%) 7 (26%) 18 (19%) 10 (30%)
 White 73 (57%) 13 (59%) 46 (59%) 14 (52%) 58 (62%) 15 (45%)
Ethnicity, no. (%)
 Latinx 16 (13%) 4 (18%) 8 (10%) 4 (15%) 0.57 12 (13%) 4 (12%) 0.92
Cumulative peanut dose at reaction (mg), mean (SD) NA NA 2343 (2743) 109 (51) <0.0001 1117 (1415)^ 1269 (1719) 0.67
Severity grade, mean (SD) NA NA 2.0 (0.8) 2.2 (1.1) 0.49 1.2 (0.8) 3.2 (0.5) <0.0001
Saliva peanut-specific IgE level (U/mL), mean (SD) 8.6 (11.1) 4.6 (5.1) 7.6 (10.9) 14.6 (13) 0.0009 7.7 (10.8) 11.2 (11.7) 0.07
Saliva peanut-specific IgA level (U/mL), mean (SD) 791.9 (1161) 1230 (2033) 683.8 (685.5) 747.8 (1267) 0.62 825.9 (1160) 695.1 (1178) 0.1
Saliva peanut-specific IgG4 level (U/mL), mean (SD) 1321 (1373) 1764 (1404) 1236 (1507) 1196 (773.8) 0.03 1382 (1273) 1154 (1629) 0.07
Serum peanut-specific IgE level (kUA/L), mean (SD) 11.0 (22.7) 6.0 (6.7) 6.9 (8.0) 26.6 (44.2) 0.0001 7.8 (9) 19.8 (41) <0.05
Peanut skin prick test (wheal size in mm), mean (SD) 8.1 (3.0) 5.3 (2.2) 8.4 (2.7) 9.4 (3.5) <0.0001 8 (3) 8.4 (2.2) 0.2
Other atopic diseases, no. (%)
 Atopic dermatitis 80 (63%) 16 (73%) 50 (64%) 14 (52%) 0.31 59 (63%) 21 (64%) 0.93
 Asthma 31 (24%) 8 (36%) 14 (18%) 9 (33%) 0.1 22 (23%) 9 (27%) 0.66
 Allergic rhinoconjunctivitis 76 (60%) 16 (73%) 46 (59%) 14 (52%) 0.32 59 (63%) 17 (52%) 0.26
Other food allergy, no. (%)
 Cow’s milk 20 (16%) 3 (14%) 12 (15%) 5 (19%) 0.89 14 (15%) 6 (18%) 0.66
 Egg 37 (29%) 5 (23%) 21 (27%) 11 (41%) 0.3 25 (27%) 12 (36%) 0.29
 Sesame 53 (42%) 8 (36%) 33 (42%) 12 (44%) 0.84 39 (41%) 14 (42%) 0.93
 Wheat 14 (11%) 5 (23%) 6 (8%) 3 (11%) 0.14 10 (11%) 4 (12%) 0.82
 Tree nut* 85 (67%) 18 (82%) 53 (68%) 14 (52%) 0.08 63 (67%) 22 (67%) 0.97
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P values were calculated using the Fisher’s exact test for categorical variables, Kruskal-Wallis or Mann-Whitney test for continuous variables

*

Almond, hazelnut, cashew, and/or walnut

^

Cumulative peanut dose for participants with reactions

NPA=Not Peanut Allergic

We first sought to determine associations between pre-challenge saliva antibody profiles and peanut allergy reaction thresholds (Fig. 1). We found that children with high pre-challenge saliva PN IgE (≥ median 1.9 U/mL) had higher odds of LT peanut allergy (OR 3.88, 95% confidence interval [CI] 1.57–9.51). This finding was concordant with results based on serum antibody levels, where participants with high serum PN IgE levels (≥ 15 kUA/) also had higher odds of LT peanut allergy (OR 6.19, 95% CI 2.11–16.24). Both saliva PN IgE (Spearman r = − 0.39, P < 0.0001) and serum PN IgE (serum PN IgE: Spearman r = − 0.46, P < 0.0001) levels were negatively correlated with cumulative peanut protein ingested (Fig. E1). Because serum PN antibody isotype ratios have previously been examined in oral immunotherapy and early peanut introduction trials,5,24,25 and saliva PN IgG4:PN IgE and PN IgA:PN IgE ratios also appear to be modulated by peanut immunotherapies,24,26 we also examined these ratios in saliva in this cohort. We found that children with high pre-challenge PN IgA:PN IgE or PN IgG4:PN IgE ratios (≥median 87.4 and 203.2, respectively) in their saliva had significantly lower odds of LT peanut allergy (OR 0.33, 95% CI 0.14–0.81, and OR 0.36, 95% CI 0.15–0.91, respectively). We did not find associations between pre-challenge saliva PN IgA, PN IgG4 levels, and threshold.

Fig. 1. Associations between low threshold (LT) peanut allergy and saliva and serum antibody levels in 127 children who underwent double-blind, placebo-controlled peanut challenges.

Fig. 1.

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LT was defined based on reaction to <300 mg cumulative peanut protein. Saliva antibody levels were dichotomized based on median levels (PN IgE: 1.9 U/mL, PN IgA: 458.6 U/mL, PN IgG4: 987.9 U/mL, PN IgA:PN IgE: 87.4, PN IgG4:PN IgE: 203.2) in the cohort. Serum PN IgE levels were dichotomized based on 15 kUA/L.23 P values are from Fisher’s exact test.

Peanut allergic reactions can range from mild to severe anaphylaxis, and non-invasive biomarkers for predicting reaction severity do not exist.5 We next assessed for associations between pre-challenge saliva PN antibody levels and reaction severity (Fig. 2). In children with high saliva PN IgE (≥ median 1.9 U/mL)) as well as high serum PN IgE (≥15 kUA/L), we observed trends toward increased likelihood for high peanut allergy severity, but these associations were not significant. In contrast, children with high saliva PN IgG4 (≥ median 987.9 U/mL) had significantly lower odds of high severity reactions during peanut challenges (OR 0.39, 95% CI 0.18–0.92). We did not observe associations between saliva PN IgA, saliva PN IgA:PN IgE, saliva PN IgG4:PN IgE, and reaction severity.

Fig. 2. Associations between high severity peanut allergic reaction and saliva and serum antibody levels in 127 children who underwent double-blind, placebo-controlled peanut challenges.

Fig. 2.

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High severity peanut allergic reaction was defined as severity score ≥3 based on a consensus severity grading system for acute allergic reactions.21 Saliva antibody levels were dichotomized based on median level (PN IgE: 1.9 U/mL, PN IgA: 458.6 U/mL, PN IgG4: 987.9 U/mL, PN IgA:PN IgE: 87.4, PN IgG4:PN IgE: 203.2) in the cohort. Serum PN IgE levels were dichotomized based on 15 kUA/L23. P values are from Fisher’s exact test.

Symptoms of peanut allergic reactions span multiple organ systems, including the respiratory, gastrointestinal, and cutaneous systems, either in combination or in an isolated manner, with respiratory symptoms typically regarded as the most clinically concerning manifestation. We next examined the hypothesis that saliva PN antibodies are associated with organ-specific symptoms during peanut oral challenge (Fig. 3). We found that children with high pre-challenge saliva PN IgE (≥ median 1.9U/mL) had significantly higher odds of respiratory symptoms during oral food challenges (OR 7.98, 95% CI 2.16–26.81), including throat tightness/itching, dry hacking/cough (> 3 minutes), and/or wheezing. Consistent with this, though with lower effect size, children with high serum PN IgE (≥ 15 kUA/L) were also more likely to experience respiratory reactions (OR 4.32, 95% CI 1.53–12.79). On the other hand, participants with high pre-challenge PN IgA:PN IgE or PN IgG4:PN IgE ratios (≥median 87.4 and 203.2, respectively) had significantly lower odds of respiratory symptoms during oral challenges (OR 0.16, 95% CI 0.05–0.58, and OR 0.24, 95% CI 0.08–0.77, respectively). We did not find associations between pre-challenge PN antibody profiles and other allergic manifestations during oral challenges, including gastrointestinal, cutaneous, and multi-system symptoms.

Fig. 3. Associations between respiratory symptoms during allergic reaction and saliva and serum antibody levels in 127 children who underwent double-blind, placebo-controlled peanut challenges.

Fig. 3.

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Respiratory symptoms included throat tightness/itching, dry hacking/cough (> 3 minutes), and/or wheezing. Saliva antibody levels were dichotomized based on median level (PN IgE: 1.9 U/mL, PN IgA: 458.6 U/mL, PN IgG4: 987.9 U/mL, PN IgA:PN IgE: 87.4, PN IgG4:PN IgE: 203.2) in the cohort. Serum PN IgE levels were dichotomized based on 15 kUA/L.23 P values are from Fisher’s exact test.

As serum PN IgE levels are a current clinical standard to aid in decision-making regarding peanut allergy management, we next examined the relationships between saliva and serum PN IgE (Fig. 4). There was modest correlation between saliva PN IgE and serum PN IgE concentrations (Pearson r=0.31, P=0.0004). A serum PN IgE level of 15 kUA/L has been used as a predictive cutoff for a reactive peanut oral food challenge.23 Notably, two distinct endotypes were observed in children with serum PN IgEs below this cutoff: low saliva PN IgE (below detection limits; serumlo/salivalo - group 1) and high saliva PN IgE (serumlo/salivahi - group 2). Similarly, two endotypes were observed in children with serum PN IgEs above the clinical cutoffs: low (serumhi/salivalo - group 3) and high (serumhi/salivahi - group 4) saliva PN IgE. In both blood analyte-defined groups, there were increased rates of LT in those with high vs. low saliva PN IgE (group 1: 9.5% and group 2: 23.3%; group 3: 28.9% and group 4: 64.3%). In the same manner, we observed increased rates of the high severity in children with high vs. low saliva PN IgE within serum PN-IgE-defined groups (group 1: 19% and group 2: 30.2%; group 3: 28.6% and group 4: 43%).

Fig. 4. Peanut allergy endotypes based on serum and saliva peanut IgE levels.

Fig. 4.

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(A): Distribution and correlation of saliva and serum PN IgE levels and grouping of individuals into peanut allergy endotypes based on saliva and serum PN IgE levels. Pearson correlation coefficient and P value are indicated for the correlation between saliva and serum PN IgE levels. The vertical dashed line represents serum PN IgE cutoff of 15 kUA/L.23 The horizontal dashed line represents lower detection limit for saliva PN IgE (< 1.9 U/mL). Peanut allergy endotypes based on antibody levels are as follows: Group 1 - serumlo/salivalo (blue), Group 2 - serumlo/salivahi (magenta), Group 3 - serumhi/salivalo (orange), Group 4 - serumhi/salivahi (red). (B): Proportion of low threshold (reaction with cumulative ingestion of <300mg peanut protein, left column) and high severity (severity score ≥ 3, right column) within each peanut allergy endotype (Groups 1–4).

DISCUSSION

In this study, we examined the association between pre-challenge saliva PN antibody levels and clinical outcomes (reaction threshold, severity, and organ-specific symptoms) in 127 children who underwent double-blind, placebo-controlled peanut challenges. We found that pre-challenge saliva PN antibody profiles are associated with differential peanut allergy reaction threshold, severity, and organ-specific manifestations. In addition, saliva PN IgE levels further distinguished reaction threshold and severity in participants stratified by serum PN IgE, suggesting endotypes.

To date, attempts to identify biomarkers of peanut allergy reaction threshold and severity have relied primarily on blood analytes. In addition, each individual marker has not been able to fully predict food challenge outcomes, pointing towards the existence of complex and multifactorial biological determinants.5 While readily accessible, saliva has not been extensively explored. We previously reported on saliva microbiota, metabolites, and immune cytokine levels associated with peanut allergy status.14 Others have shown that saliva PN antibodies are modulated by sublingual and oral immunotherapies.16,26 This study expands our understanding of the oral environment in food allergy by expanding the scope of study to saliva antibody profiles and the key clinical parameters of peanut allergy reaction threshold, severity, and organ-specific symptoms. While prior work has shown that BAT can be applied to identify severe reactors and those with low threshold allergy in the research setting,5 the need for fresh blood, basophil viability limitations, analytical interferences, and varied experimental conditions remain significant barriers for clinical implementation of BAT.13,27 Saliva antibodies are stable and straightforward to measure, requiring no time-sensitive or complicated processing. In addition, saliva collection is very well-tolerated in children.14,28 Our results highlight the potential of saliva profiling as a non-invasive biomarker to aid in the risk-stratification of food allergy reaction threshold and severity, especially if further study validates its performance as a stand-alone biomarker.

Our findings from saliva PN antibody profiles paralleled observations from blood biomarker studies of peanut allergy. These prior findings in blood include the: (1) association between serum PN IgE and PN IgG4:PN IgE ratio in LT children, and (2) link between serum PN IgG4 and peanut allergy severity.5,29,30 Our results indicate the biologic relevance and predictive potential of saliva PN antibodies in peanut allergy - in particular, that they are already present in the pre-challenge state and are associated with outcomes during subsequent challenge. A role for saliva PN antibodies is also clinically supported, given that many peanut allergy reactions frequently occur within minutes, prior to systemic absorption via the lower GI tract.

Notably, we found that saliva PN IgE levels are reflective of the likelihood for respiratory manifestations during peanut challenges, with an effect size greater than that of serum PN IgE. This observation may reflect anatomical proximity between these two mucosal surfaces, or may suggest a deeper link between oral and airway immune milieu that may be of biological importance.31 Based on saliva PN IgEs, we also observed distinct and clinically relevant endotypes within patient groups categorized by current serum PN IgE clinical cutoffs. This finding suggests that saliva PN antibody measurements may provide an additional dimension of risk information when assessing pediatric candidates for peanut oral challenges. An independent cohort will be needed to validate this observation.

Various mechanisms of action for non-IgE PN antibody isotypes have previously been proposed. For IgG, these include preventing mast cell/basophil activation by direct steric blockade of antigens and mixed IgE/IgG-receptor cross-linking followed by signaling through inhibitory IgG FC receptor (FCγRIIb).17,18 For IgA, it is thought to contribute to antigenic exclusion and has been shown to be protective against anaphylaxis in murine models,19 although the role of mucosal IgA has not been directly studied in the context of food allergy reaction threshold, severity, and organ-specific symptoms in human subjects. Previously, saliva IgE was reported to be below the detection limit in most healthy subjects, but its prevalence was higher in atopic individuals.32,33 Further investigation is required to determine whether saliva antibody compositions are biomarkers and/or direct biologic determinants of peanut allergy reaction threshold, severity, and organ-specific symptoms.

Our study has limitations. While our examination of 127 children is large for a study of this kind, particularly since double-blind, placebo-controlled peanut challenges were conducted for each participant, a larger cohort will be needed to thoroughly test the clinical performance and relevant cutoffs of saliva antibody levels for peanut allergy reaction threshold, severity, and organ-specific symptom assessment - either on their own or in combination with other biomarkers. The study was conducted at an academic center where many food allergic patients are referred, and thus it is possible that the cohort included participants with more complex peanut allergy than in the broader community. Future studies examining the natural history of saliva PN antibodies over time may also provide important insights on the clinical utility of these biomarkers. As a cross-sectional human biomarker study, future studies utilizing murine models and/or human-derived cells/tissues could be performed to dissect the mechanisms underlying our findings, especially regarding the biological roles of mucosal allergen-specific antibody isotypes. Despite these limitations, the ease of saliva collection, lack of need for time-dependent processing, and relative ease of ELISA-based measurement make saliva antibody levels an intriguing biomarker candidate.

In summary, we identified saliva antibody profiles associated with reaction threshold, severity, and organ-specific symptoms during double-blind, placebo-controlled peanut challenges. Additionally, our data suggest that saliva antibodies provide further insight beyond serum levels frequently used in clinical practice. These findings raise the potential for non-invasive saliva biomarkers that could be used to assess reaction threshold, severity, and organ-specific symptoms in peanut allergy, providing tools for real-world clinical needs in food allergy management. Further dissecting the biological effects of mucosal PN antibodies could also provide mechanistic insights on the biological determinants that govern peanut allergy reaction threshold and severity.

Supplementary Material

Supp.Materials

Key Messages.

  • Reaction threshold, severity, and symptoms vary widely across peanut allergic individuals. There is a lack of non–invasive markers to aid in risk-stratification.

  • We found that pre-challenge levels of peanut antibodies in saliva were associated with reaction threshold, severity, and organ-specific manifestations in peanut allergic children. Saliva profiles revealed distinct endotypes among peanut allergic children.

  • Saliva antibody levels have the potential to aid in risk-stratification of peanut allergy reaction threshold and severity.

Acknowledgments

This study was funded by the National Institutes of Health grants R01AI147028 and U19AI136053. We thank the children and their families for their participation in this research as well as the nursing and support staff for sharing in the care of these participants. We would also like to thank Drs. Michael Kulis, Cecilia Berin, and Alexander Grishin for their thoughtful guidance on protocols.

Funding:

National Institutes of Health grants R01AI147028 and U19AI136053.

Conflicts of interest:

SS reports royalty payments from UpToDate and from Johns Hopkins University Press; grants to his institution from the National Institute of Allergy and Infectious Diseases, from Food Allergy Research and Education, and from Pfizer; and personal fees from the American Academy of Allergy, Asthma and Immunology as Deputy Editor of the Journal of Allergy and Clinical Immunology: In Practice, outside of the submitted work.

Abbreviations:

BAT

Basophil activation test

PN

peanut

SPT

skin prick test

Footnotes

All other authors report no competing interests.

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