Antigenic Liposomes for Generation of Disease-specific Antibodies

Bednar, Kyle J.; Hardy, Lakeya; Smeekens, Johanna M.; Raghuwanshi, Dharmendra; Duan, Shiteng; Kulis, Mike; Macauley, Matthew

doi:10.3791/58285

Cited by 14 publications

(11 citation statements)

References 27 publications

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“…Peanut Extract- Peanut extract was prepared by extracting peanut protein from roasted defatted peanut flour (Golden Peanut, Alpharetta, GA) as previously described ( 37 ).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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Background10% of US residents have food allergies, including 2% with peanut allergy. Mast cell mediators released during the allergy effector phase drive allergic reactions. Therefore, targeting sensitized mast cells may prevent food allergy symptoms.ObjectiveWe used novel, human, allergen-specific, IgE monoclonal antibodies (mAbs) created using human hybridoma techniques to design an in vitro system to evaluate potential therapeutics targeting sensitized effector cells.MethodsTwo human IgE mAbs specific for peanut, generated through human hybridoma techniques, were used to sensitize rat basophilic leukemia (RBL) SX-38 cells expressing the human IgE receptor (FcϵRI). Beta-hexosaminidase release (a marker of degranulation), cytokine production, and phosphorylation of signal transduction proteins downstream of FcϵRI were measured after stimulation with peanut. Degranulation was also measured after engaging inhibitory receptors CD300a and Siglec-8.ResultsPeanut-specific human IgE mAbs bound FcϵRI, triggering degranulation after stimulation with peanut in RBL SX-38 cells. Sensitized RBL SX-38 cells stimulated with peanut increased levels of phosphorylated SYK and ERK, signal transduction proteins downstream of FcϵRI. Engaging inhibitory cell surface receptors CD300a or Siglec-8 blunted peanut-specific activation.ConclusionAllergen-specific human IgE mAbs, expressed from human hybridomas and specific for a clinically relevant food allergen, passively sensitize allergy effector cells central to the in vitro models of the effector phase of food allergy. Peanut reproducibly activates and induces degranulation of RBL SX-38 cells sensitized with peanut-specific human IgE mAbs. This system provides a unique screening tool to assess the efficacy of therapeutics that target allergy effector cells and inhibit food allergen-induced effector cell activation.

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“…Peanut Extract- Peanut extract was prepared by extracting peanut protein from roasted defatted peanut flour (Golden Peanut, Alpharetta, GA) as previously described ( 37 ).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2022

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“…Protein extractions were performed as reported previously ( 21 ). Briefly, proteins were extracted from roasted, defatted peanut flour (Golden Peanut, Alpharetta, GA), roasted, defatted walnut flour (Holmquist Hazelnut Orchards, Lynden, WA), nonfat dry milk powder (The Milky Whey, Missoula, MT) or egg white powder (Deb El Foods, Elizabethport, NJ) in PBS (supplemented with 1 M NaCl for peanut and walnut extractions).…”

Section: Methodsmentioning

confidence: 99%

“…Antigen-specific IgE, IgG1 and IgG2a were quantified by ELISA as described previously ( 21 ). Briefly, 96-well plates were coated with 20 µg/ml peanut, walnut, milk, or egg extract for samples, or 20 µg/ml HSA-DNP (Millipore Sigma, St. Louis, MO) for standard curves, and blocked with 2% BSA in PBS-0.5% Tween.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2021

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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.

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“…Peanut allergen extract was prepared from roasted de‐fatted peanut flour (Golden Peanut) in PBS with 1 M NaCl as described previously . Extracts of indoor dust (ID) were prepared as previously described .…”

Section: Methodsmentioning

confidence: 99%

“…Peanut allergen extract was prepared from roasted de-fatted peanut flour (Golden Peanut) in PBS with 1 M NaCl as described previously. 46 Extracts of indoor dust (ID) were prepared as previously described. 47 Briefly, vacuumed dust samples were collected from a single-family residence over several months to minimize sampling variability.…”

Section: Reagentsmentioning

confidence: 99%

Indoor dust acts as an adjuvant to promote sensitization to peanut through the airway

Smeekens

Immormino

Balogh

et al. 2019

Clin Experimental Allergy

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Background There is growing evidence that environmental peanut exposure through non‐oral routes, including the skin and respiratory tract, can result in peanut sensitization. Environmental adjuvants in indoor dust can promote sensitization to inhaled antigens, but whether they contribute to peanut allergy development is unclear. Objective We investigated whether indoor dust promotes airway sensitization to peanut and peanut allergy development in mice. Methods Female and male C57BL/6J mice were exposed via the airways to peanut, indoor dust extract, or both for 2 weeks. Mice were then challenged with peanut and assessed for anaphylaxis. Peanut‐specific immunoglobulins, peanut uptake by lung conventional dendritic cells (cDCs), lung innate cytokines, and T cell differentiation in lung‐draining lymph nodes were quantified. Innate cytokine production by primary human bronchial epithelial cells exposed to indoor dust was also determined. Results Inhalational exposure to low levels of peanut in combination with indoor dust, but neither alone, resulted in production of peanut‐specific IgE and development of anaphylaxis upon peanut challenge. Indoor dust triggered production of innate cytokines in murine lungs and in primary human bronchial epithelial cells. Additionally, inhaled indoor dust stimulated maturation and migration of peanut‐laden lung type 1 cDCs to draining lymph nodes. Inhalational exposure to peanut and indoor dust induced peanut‐specific T helper 2 cell differentiation and accumulation of T follicular helper cells in draining lymph nodes, which were associated with increased B cell numbers and peanut‐specific immunoglobulin production. Conclusions & clinical relevance Indoor dust promotes airway sensitization to peanut and development of peanut allergy in mice. Our findings suggest that environmental adjuvants in indoor dust may be determinants of peanut allergy development in children.

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Antigenic Liposomes for Generation of Disease-specific Antibodies

Cited by 14 publications

References 27 publications

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

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

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

Indoor dust acts as an adjuvant to promote sensitization to peanut through the airway

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