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.

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.

Fecal IgA, Antigen Absorption, and Gut Microbiome Composition Are Associated With Food Antigen Sensitization in Genetically Susceptible Mice.

Food allergy is a potentially fatal disease affecting 8% of children and has become increasingly common in the past two decades. Despite the prevalence and severe nature of the disease, the mechanisms underlying sensitization remain to be further elucidated. The Collaborative Cross is a genetically diverse panel of inbred mice that were specifically developed to study the influence of genetics on complex diseases. Using this panel of mouse strains, we previously 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 associated with sensitization in CC027/GeniUnc mice following oral exposure to peanut, walnut, milk, or egg. 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. Naïve CC027/GeniUnc mice had markedly lower total fecal IgA compared to C3H/HeJ, which was accompanied by stark differences in gut microbiome composition. Sensitized CC027/GeniUnc mice had significantly fewer CD3+ T cells but higher numbers of CXCR5+ B cells and T follicular helper cells in the mesenteric lymph nodes compared to C3H/HeJ mice, which is consistent with their relative immunoglobulin production. After oral challenge to the corresponding food, peanut- and walnut-sensitized CC027/GeniUnc mice experienced anaphylaxis, whereas mice exposed to milk and egg did not. Ara h 2 was detected in serum collected post-challenge from peanut-sensitized mice, indicating increased absorption of this allergen, while Bos d 5 and Gal d 2 were not detected in mice exposed to milk and egg, respectively. Machine learning on the change in gut microbiome composition as a result of food protein exposure identified a unique signature in CC027/GeniUnc mice that experienced anaphylaxis, including the depletion of Akkermansia. Overall, these results demonstrate several factors associated with enteral sensitization in CC027/GeniUnc mice, including diminished total fecal IgA, increased allergen absorption and altered gut microbiome composition. Furthermore, peanuts and tree nuts may have inherent properties distinct from milk and eggs that contribute to allergy.