Engineering Nanoparticle Therapeutics for Food Allergy

Abstract

Food allergy is a growing public health issue among children and adults that can lead to life-threatening anaphylaxis upon allergen exposure. The gold standard for disease management includes food avoidance and emergency epinephrine administration because current allergen-specific immunotherapy treatments are limited by adverse events and unstained desensitization. A promising approach to remedy these shortcomings is the use of nanoparticle-based therapies that disrupt disease-driving immune mechanisms and induce more sustained tolerogenic immune pathways. The pathophysiology of food allergy includes multifaceted interactions between effector immune cells, including lymphocytes, antigen presenting cells, mast cells, and basophils, mainly characterized by a T helper cell type 2 (Th2) response. Regulatory T cells, T helper cell type 1 (Th1) responses, and suppression of other major allergic effector cells have been found to be major drivers of beneficial outcomes in these nanoparticle therapies. Engineered nanoparticle formulations that have shown efficacy at reducing allergic responses and have uncovered new mechanisms of tolerance include polymeric-, lipid-, and emulsion-based nanotherapeutics. This review highlights the recent engineering design, mechanisms induced, and future potential therapeutic targets of these nanoparticles.

Introduction:

Food allergy is a growing global health concern, affecting an estimated 10% of adults and 8% of children [1,2]. The substantial economic and quality of life impact of the disease are also increasing, highlighting the need for the development of novel therapeutics [3,4]. The pathology of food allergy is driven by a poorly controlled abnormal immune response to otherwise innocuous dietary antigens, resulting in a wide range of clinical manifestations, from mild cutaneous symptoms to severe life-threatening anaphylaxis [5]. Despite recent advances in the development of therapies for food allergies, the primary options for patients remain allergen avoidance and upon accidental allergen exposure, use of medications, such as epinephrine for emergency use during life-threatening reactions and antihistamines for managing mild allergic reactions [6].

The most well-studied approach for the suppression of food allergies is allergen-specific immunotherapy (AIT). AIT involves the exposure of a patient to increasing doses of allergen with the goal of desensitizing the immune system to that allergen. This approach includes the only FDA approved treatment to prevent allergic food reactions, Palforzia^®, which is an oral immunotherapy (OIT) product for peanut allergy [7]. While OIT is the most studied route of AIT for food allergy, other routes of administration are under development, including epicutaneous (EPIT), sublingual (SLIT) and subcutaneous (SCIT) [8–10].

While the induction of immunological tolerance to the allergen is the ultimate goal, most AITs for food allergy have instead induced desensitization [11]. Patients often are only able to tolerate allergen while they remain on the therapy, but frequently lose therapeutic benefit within weeks of stopping the treatment [7,12]. In addition to not producing durable immune tolerance, AIT protocols mostly require extended protocols of allergen up-dosing to reach the maintenance dose and may require months to years at the maintenance dose to achieve significant desensitization. Thus, novel therapeutics capable of inducing durable tolerance more quickly and with fewer required doses are being pursued [13]. This review will provide an overview of the development of novel nanoparticle-based therapeutics for enhancing efficacy in AIT and describe the mechanisms by which these therapies can be harnessed for tolerance induction.

Food allergy pathology:

The development of food allergy involves complex interactions between multiple types of immune cells, including CD4⁺ T helper cells (Th cells), mast cells, basophils, antigen presenting cells (APCs), such as dendritic cells (DCs) and B cells. Allergic sensitization is thought to be initiated at an epithelial surface, such as the gastrointestinal tract, airway or skin, where APCs take up and process antigen for presentation, leading to activation of naïve CD4⁺ T cells [14]. Upon exposure to allergen, epithelial cells and innate lymphoid cells (ILCs) release a subset of pro-inflammatory cytokines termed alarmins (interleukin-25 (IL-25), IL-33 and thymic stromal lymphopoietin (TSLP)) [15–17]. Alarmins drive the development of APCs that promote the differentiation of naïve CD4⁺T cells into Th2 cells that orchestrate the immunological responses that drive allergic reactivity, including inflammatory cell recruitment, mast cell activation, and mucus production [18]. Th2 cells release cytokines (IL-4, IL-5, IL-9, and IL-13) that play multiple roles in the pathology of food allergy [19]. IL-5 recruits and activates eosinophils, contributing to tissue inflammation, while IL-13 promotes mucus production and alters epithelial barrier integrity, enhancing allergen penetration and amplifying the allergic response. IL-4 and IL-5 promote the class-switching of B cells to produce allergen-specific IgE antibodies, which is required for sensitization to the allergen.

Food allergy can be non-IgE- or IgE-mediated. While non-IgE-mediated food allergy still poorly understood, it has been described that its mechanisms are mainly mediated by a T cell involvement [20,21]. On the other hand, IgE-mediated food allergy accounts for most allergic reactions to foods [22]. Allergen-specific IgE binds to high affinity receptors (FcɛR1) on mast cells and basophils. Subsequent allergen exposure leads to crosslinking of IgE antibodies on the cell surface of sensitized mast cells and basophils, inducing degranulation and the release of preformed inflammatory mediators, such as histamine, prostaglandins, leukotrienes, and cytokines, which initiate the allergic cascade [23]. These mediators cause vasodilation, increased vascular permeability, smooth muscle contraction, and recruitment of inflammatory cells, resulting in the characteristic clinical symptoms of food allergy [22]. The resulting symptoms may be mild, including edema, urticaria, emesis, and diarrhea, or severe such as respiratory arrest, cardiovascular collapse, hypotension, and shock. Due to the central role of Th2-derived cytokines and IgE in food allergy, the inhibition of these pathways is a target for the development of therapeutics to treat established disease.

Mechanisms of allergen-specific immune tolerance:

The primary effector molecule derived from immune cells in allergy is allergen-specific IgE, and anti-IgE treatment is currently reserved for selected patient [24]. The production of both high affinity antibody critical for host defense against infection and the generation of high-affinity allergen-specific IgE are driven by similar mechanisms, i.e., the antigen-specific generation of germinal centers, the cross-presentation of antigenic peptides by B cells to CD4⁺ T cells, and the induction of B cell Ig class-switching. During T cell-dependent antibody responses, B cells present antigenic peptide complexed with major histocompatibility complex class II (MHC II) to antigen-specific CD4⁺ T cells inside the T cell zone of the primary follicles within secondary lymphoid tissues, i.e., the spleen or lymph nodes, or immune follicles present within the lamina propria of mucosal tissue. As a result of cognate TCR ligation and costimulatory signaling by the CD4⁺ T cells and the B cells, respectively, B cells proliferate and germinal centers form, with the eventual outcome of increased affinity of the antigen-specific antibodies and isotype class-switching, which is aided by T follicular helper (Tfh) cells [25,26]. While T cells of various phenotypes have immunosuppressive properties throughout the body as well as within germinal centers, the most well characterized at present are CD4⁺CD25⁺ Forkhead box P3⁺ (FoxP3) T regulatory cells (Tregs). Tregs can migrate into B cell follicles and directly regulate germinal center responses [27,28].

Tregs are critical in maintaining tolerance to self-antigens and allergens [29]. Tregs can be generated in the thymus to mediate tolerance to self-antigens (natural; nTregs) or be induced in the periphery (iTregs) after non-self-antigen or allergen exposure in the presence of molecules such as TGF-β (transforming growth factor-β), retinoic acid (RA), and IL-2, which are constantly secreted by APCs in the lungs [30,31] and gastrointestinal tissues [32–35], and by activated T cells within the local microenvironment. Suppression of immune responses by Tregs is mediated by both contact-dependent and contact-independent mechanisms. For example, Tregs express high levels of the high affinity IL-2 receptor molecule, CD25, and thereby sequester IL-2 from effector CD4⁺ T cells. Tregs also express cell surface co-inhibitory molecules such as cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1) and lymphocyte-activation gene 3 (LAG-3) and secrete regulatory cytokines (TGF-β and IL-10), as well as cytotoxic molecules (granzymes and perforin). Collectively, these regulatory mechanisms mediate Treg functional activity reviewed in [36,37].

The importance of Tregs in allergy is well known [38,39], and mutations in the principal transcription factor, FoxP3 [40], or the regulatory molecules CD25, CTLA-4 or IL-10 [41–43], not only lead to multi-organ failure, associated with several autoimmune disorders (Immune Dysregulation Polyendocrinophaty, Enterophaty X-linked syndrome;IPEX), but usually accompany the development of serious allergic symptoms (X-linked autoimmunity-allergic dysregulation; XLAAD) [44,45], characterized by a Th2 cell phenotype, eosinophilia, and IgE-hyperglobulinemia [46]. The allergen-associated characteristics are also present in scurfy mice (mutation in the FoxP3 gene) [47], and in vivo Treg depleted mice failed to control allergen-specific responses [48–50], which are mediated in part by IL-10 secretion [51]. Notably, several studies have described that Tregs [52–55] and CD4⁺ IL-10-secreting type 1 regulatory T cells (Tr1) [53,56,57] are significantly reduced and express a less stable phenotype in allergy patients [53,58,59]. In support of a functional role for Tregs for control of allergic responses, passive transfer of Treg cells into sensitized mice decreases allergen-specific immune responses [60,61]. Targets of current therapies for allergic disease include Treg suppressive mechanisms, which have been shown to modulate several pathogenic cells involved in the disease: Th2 cells and ILC2 cells, mast cells, basophils, and B cells. Tregs inhibit the activation of Th2 cells and the secretion of their effector allergic cytokines (i.e., IL-4, IL-5, and IL-13) needed for mast cell activation and allergen IgE class-switching. It has also been shown that Treg cells can suppress ILC2 cells [62,63], which secrete large amounts of IL-5 and IL-13 [64]. Importantly, Treg cells can directly interact with mast cells via OX40-OX40L interaction [65] and regulatory cytokines, IL-10 and TGF-β [66], thereby inhibiting mast cell degranulation. Therefore, the development of new therapies that can induce allergen-specific Tregs and regulatory molecules to re-establish/induce long-term tolerance in patients is a priority focus within the field.

Nanoparticles as an AIT for allergy:

As mentioned above, currently approved therapies for the treatment of food allergy are limited. A putative therapeutic strategy being examined is the utilization of nanoparticle-based therapeutics. Nanoparticles are generally defined to be between 1 and 100 nm in diameter. However, this review will include any submicron particle, as the physiologically relevant scale to interface with the immune system as an antigen-specific immunotherapy has been found to be in the range from 100 to 600 nm [78]. This review will focus on new mechanistic insights explaining how engineered nanoparticles exert therapeutic effects in allergic diseases as summarized in Table 1. The engineered nanoparticles of specific focus include polymeric nanoparticles, lipid nanoparticles, and nanoemulsions (Figure 1A). For a more comprehensive review of nanoparticle design considerations, please refer to Mitchell et al. [79] and Banik et al. [80] as well as for nanoparticle designs used in allergy, please refer to Pohlit et al. [81], Longo et al. [82], and Paris et al. [83].

Table 1.

Nanoparticle Therapeutics in Food Allergy

Disease		Study Subject	Study Design	Nanoparticle System	Antigen Delivered	Route of Nanoparticle Delivery and Dosing Regime	Results	Citation
Shrimp allergy	Mouse	i.g. recombinant arginine kinase (AK)/cholera toxin sensitization, i.g. AK challenge		PLGA-PVA, 195.5 nm	Encapsulated CpG and AK-specific T cell epitope peptide AK81–100 consists of 20 amino acids in length	i.p. daily doses delivering 25 μg AKp and 100 μg CpG-ODN for 6 days after sensitization	- Reduced incidence of diarrhea and hypothermia - Lower levels of allergen-specific IgE and increased IgG2a in serum - Th2 cell cytokines suppression and Th1 cytokines induction - Increased FoxP3 and IL-10 protein expression and decreased STAT6 and GATA3 expression in the small intestine	[67]
Cow’s milk allergy	Mouse	i.g. whey/cholera toxin sensitization, oral or intradermal challenge with whey		PLGA-PVA, 242 nm, −0.8 mV	Encapsulated CpG and an 18 amino acid β-lactoglobulin (BLG)-specific T cell epitope peptide derived from the chain B of bovine β-lactoglobulin	Oral prophylactic treatment of six daily oral doses of nanoparticles delivering 160 μg BLG peptide and 3 μg CpG in approx. 20 mg of nanoparticle	- Prevented the whey-induced allergic skin reactivity and associated rise in serum BLG-specific IgE, but not efficacious for allergic reactions after oral challenge - Increased PD-L1+ DCs in the spleen but decreased in the mesenteric lymph nodes - Increased Treg/Th2 cell and Th1/Th2 cell ratios in the spleen	[68]
Peanut allergy	Mouse	i.p. peanut/alum sensitized mice, oral challenge with peanut extract		PLGA-PEMA, 563.5 nm, −61.2 mV	Peanut extract 25.75 ug per dose	Three i.v. doses of 2.5 mg of nanoparticles over a 15-day period after sensitization	- Reduced anaphylaxis-related hypothermia and mast cell degranulation - Broad suppression of splenic Th1-, Th17-, Th2-, Treg- associated cytokines	[69]
Cow’s milk allergy	Mouse	i.p. alum/casein induced allergy, i.p. or oral challenge with casein		Soybean oil with cetyl pyridinium chloride, Tween 80 and ethanol in water nanoemulsion, 350–400 nm	20 μg of casein	Four intranasal doses: one 12 μL dose every four weeks after sensitization	- Decrease in anaphylaxis related hypothermia and mast cell degranulation - 20-fold increase in cow’s milk specific IgG2a titers, but cow’s milk specific IgE titers were still elevated - Decreased mast cell frequency in the small intestine - Suppression of Th2 cytokines in the MLN, protection transferred to naïve mice by serum transfer	[70]
Peanut and egg allergy	Mouse	i.p. alum/peanut extract and ovalbumin (OVA) induced allergy, oral challenge with OVA and peanut extract		Soybean oil with cetyl pyridinium chloride, Tween 80 and ethanol in water nanoemulsion, 350–400 nm	20 μg OVA and 20 μg peanut extract	Three intranasal doses: one 12 μL dose every four weeks after sensitization	- Suppression of Th2-polarized immune responses, alarmins and ILC2 - Nanoemulsion-induced bystander suppression of reactivity required IFN-γ and the presence of an allergen in the NE vaccine.	[71]
Peanut allergy	Mouse	Dysbiosis induced by i.g. vancomycin before sensitization, oral peanut extract/cholera toxin induced allergy, i.p. challenge with peanut extract		Neutral polymeric micelles: −0.34 mV and 44.7 nm, and negatively charged polymeric micelles: −31.5 mV and 39.9 nm	Butyrate	i.g. 400 mg/kg twice daily for two weeks after sensitization	- Reduced anaphylaxis-related hypothermia, mast cell degranulation, and serum peanut-specific IgE and peanut-specific IgG1 titers - Did not affect Treg populations - Lead to downregulation of MHC-II and CD86 on myeloid cells	[72]
Peanut allergy	Mouse	i.v. adoptive transfer of splenocytes from peanut extract/cholera toxin sensitized mice, i.p. challenge with peanut extract		PEG-DSPE liposomal nanoparticles, 120 nm	200 μg of Ara h 1, Ara h 2, or Ara h 3 and high-affinity CD22 ligand	two i.v. 300 μmol/mL or 600 μmol/mL doses 14 days apart after sensitization before challenge	- Reduced allergen-specific IgE, IgG1, anaphylaxis-associated hypothermia - Unresponsiveness sustained for at least 3 months	[73]
Passive anaphylaxis	Mouse	i.v. or intradermal passive anaphylaxis by anti-TNP-IgE, i.v. challenge with TNP-liposome		PEG-DSPE liposomal nanoparticles	TNP and Siglec-8 ligand	One i.v. dose ranging from 50 μg to 159 μg after sensitization	- Reduction in anaphylaxis-induced hypothermia - Reduced IL-6 release by mast cells	[74]
Peanut allergy	Mouse	i.g. peanut extract/cholera toxin induced allergy, i.p. challenge with peanut extract		Liver-targeting lipid nanoparticle surface decorated with mannose ligand , 148.7 nm, −4.33mV	mRNA for a 15-mer MHC-II binding epitope of the dominat peanut allergen, Ara h2	Two i.v. 1.25 mg/kg doses 1 week a part prophylactic ally and post-sensitization	- Suppression of Th2-mediated cytokine production, IgE synthesis, and mast cell release - Increased IL-10 and TGF-β production in the peritoneum	[75]
Peanut allergy	Human	Human peanut allergic patients, Double-Blind, Placebo-Controlled Food Challenge		Negatively charged PLGA nanoparticles	Purified peanut extract drug substance	i.v. 1mg to 8mg dose at 5 μg of purified peanut extract per mg of PLGA on days 1 and 8	TBD	[76]
Celiac disease	Human	Celiac disease patients, 14-day oral gluten challenge (12 g/d of gluten for 3 days followed by 6 g/d for 11 days beginning 7 days after the second infusion of treatment)		PLGA-surfactants, 489 nm, −45 mV	8.4 μg of deamidated and native gliadin per mg of PLGA	8 mg/kg, up to a maximum of 650 mg on days 1 and 8, via a 30-minute intravenous infusion	- No adverse events due to treatment occurred - 88% reduction in change from baseline in interferon-γ spotforming units vs placebo - Reduction of α4β7+CD4+ , αEβ7+CD8, and γδ effector memory T cells	[77]

Figure 1. Engineered nanoparticles design considerations and route of delivery.

Engineered nanoparticles used in the past 3 years for allergen-specific immunotherapy have included polymeric-, lipid-, and nanoemulsion-based designs (A). Changing the components of these nanoparticle changes their targeted effect. The chosen polymers, lipids, and surfactants all contribute to the size, surface charge, and therapeutic immune mechanism. These components can include functional groups used to chemically bond allergen proteins, peptides, and other disease-modifying molecules to the nanoparticles. Additionally, these nanoparticles can encapsulate molecules that need to move through the immune system “stealthily” without causing adverse allergic reactions. To better target specific immune populations, different routes of administration can be used including intravenous, intranasal, oral, and intraperitoneal injections (B).

Nanoparticles are suggested as an advantageous approach to AIT as compared to oral-, sublingual-, subcutaneous-, and epicutaneous-based AIT therapies, because nanoparticles may act as masked allergen transports, thereby minimizing allergen degradation and anaphylactic responses, allowing for routes of delivery that better target immune cells of interest without adverse event risk (Figure 1B). Additionally, nanoparticles can be engineered to target specific immune cells for maximal tolerogenic benefit. Nanoparticles interface with the immune system as mimics of apoptotic cell debris or pathogens based on their size and surface potential [78,84]. In the context of allergy, nanoparticles have been shown to induce Tregs, inactivate pathogenic T cells by acting on APCs communicating with T cells, and skew allergy-driving Th2 cell responses towards more allergen-tolerating Th1 cell responses, as characterized by CD4⁺ T cell secretion of interferon-gamma (IFN-γ) and B cell class-switching to IgG2 (Figure 2).

Figure 2. Allergic disease-associated mediators and nanoparticle modulation of these cellular mechanisms.

The present figure illustrates the cellular outcomes following allergen-specific nanoparticle (as shown by the blue arrows), which consequently decrease allergic disease-associated immune pathways (as shown by the red arrows). Engineered nanoparticles containing allergen regulate immune responses in food allergy by regulation of co-inhibitory molecules in antigen presenting cells (APCs) (A), which directly inhibit activation of Th2 and effector T cells in diseases (B), modulating the balance of Th1 vs Th2 cells responses (C), affected by the increase of T regulatory FoxP3⁺ (Treg) and IL-10-producing type 1 regulatory T cells (Tr1) (D). Globally, these mechanisms directly affect hyperglobulinemia, inducing tolerance in Th2 cells and/or a switch from IgE- to IgG2-dominant responses (E).

Poly(lactic-co-glycolic acid) nanoparticles:

Poly(lactic-co-glycolic acid) (PLGA) has been widely used for nanoparticle fabrication due to extensive applications in FDA-approved biologics, biodegradability, controlled release of encapsulated drugs and antigens, and ease of manufacturing [85]. Recent allergen-specific PLGA nanoparticle designs include adjuvants either decorating the surface or encapsulated within these nanoparticles to allow for use with less immunogenic molecules, such as smaller T cell epitopes of allergen as opposed to full protein. This approach improves the safety profile of these therapeutics since short peptides cannot crosslink IgE bound to receptors on allergy effector cells to lead to adverse events during treatment. However, these epitopes are known to elicit only a weak immune response and require an adjuvant to exert a tolerogenic immune response. One such study in a mouse model of shrimp allergy used PLGA nanoparticles that co-encapsulated a 20 amino acid T cell epitope of the shrimp allergen arginine kinase (AKp) with TLR9 agonist CpG-ODN [67]. PLGA nanoparticles of 195.5 nm size were fabricated using a solvent evaporation double emulsion method with polyvinyl alcohol (PVA) as the emulsifier and were given daily doses intraperitoneally delivering 25 μg AKp and 100 μg CpG-ODN for 6 days after an oral cholera toxin induced sensitization to recombinant arginine kinase (AK), a major shrimp allergen, followed by intragastric AK challenge. The therapeutic efficacy of these CpG-AKp nanoparticles was characterized by reduced diarrhea incidence and hypothermia after challenge. Lower levels of serum AK-specific IgE and IgG1 and the induction of AK-specific IgG2a, suggests skewing towards Th1 cell phenotypes, as confirmed by increased IFN-γ and decreased IL-4 and IL-13 by splenocyte recall assay. Increased expression of FoxP3 and IL-10 in the small intestine and decreased expression of Th2 cell transcription factors, STAT6 and GATA3, suggest Tregs and decreased Th2 cells are responsible for the therapeutic benefit.

Another study in cow’s milk allergy also used a similar approach of encapsulating an 18 amino acid β-lactoglobulin (BLG)-specific T cell epitope with CpG in 242 nm, −0.8 mV PLGA nanoparticles [68]. This study used a prophylactic model such that 6 daily oral doses of nanoparticles co-encapsulating 160 μg BLG peptide and 3 μg CpG was given before oral whey/cholera toxin sensitization, followed by intradermal or oral whey exposure. The efficacy at reducing intradermal allergic reactivity was attributed to reduced BLG-specific IgE. However, upon oral allergen exposure, the nanoparticle treatment group was not significantly different from the untreated group as characterized by body temperature and shock scores. Still, the treatment increased Treg/Th2 cell and Th1/Th2 cell ratios in the spleen as well as IFN-γ. This result may be explained by an increase tolerogenic splenic DCs, defined by lower CD80/86 and MHC II expression and increased PD-L1 expression. However, these changes were not accompanied by Th2 cell cytokine suppression or changes in allergen-specific IgG1 or IgG2 levels, which may account for the maintained allergic reactivity after oral challenge and mast cell degranulation after intradermal challenge. Delivering a combination of nanoparticles delivering either CpG or BLG peptide together did not show tolerogenic splenic DC induction, suggesting the TLR9-agonist effects of CpG alongside the antigen presentation of BLG peptide in MHC II within the same DC is required for tolerogenic DC phenotype skewing.

PLGA particles that do not include a specific adjuvant have successfully achieved protective responses, such as Hughes et al. treating peanut/alum sensitized mice with 3 intravenous doses of 2.5 mg of PLGA nanoparticles encapsulating 25.75 ug of peanut extract over a 15-day period to provide protection against oral peanut challenge, as characterized by reduced anaphylaxis-related hypothermia and mast cell degranulation [69]. This outcome could be attributed to combinatorial effect of intravenous delivery, the larger ~550 nm size and poly(ethylene-alt-maleic anhydride) (PEMA) surfactant stabilizer preferentially targeting APCs in the liver and spleen. This study did not detect a Th1 cell phenotype skewing, but rather a broad suppression of splenic Th1-, Th17-, Th2-, and Treg-associated cytokines, suggesting potentially a more tolerogenic outcome rather than immune phenotype skewing seen in other nanoparticle treatments.

Among the novel nanoparticle therapies under development for the treatment of allergy, the utilization of intravenously administered antigen-containing carboxylated PLGA nanoparticles has been shown to be efficacious in experimental models of both autoimmune disease and allergy [86]. Data from these studies show that intravenous antigen-specific PLGA nanoparticle treatment induces a significant increase in both FoxP3⁺ Tregs and IL-10⁺ CD4⁺ Tr1 cells. Additionally, treatment induces an increase in the level of CTLA-4 and PD-1 expressed, which has subsequently been shown to be required for antigen-specific PLGA nanoparticle treatment function [87]. These pre-clinical studies have culminated in the antigen-specific PLGA nanoparticle first in human clinical studies with intravenous TAK-101/TIMP-GLIA and demonstrated successful induction of antigen-specific tolerance in celiac patients orally challenged with gluten [77]. These studies assessed safety, tolerability, pharmacokinetics, and efficacy of TAK-101/TIMP-GLIA treatment to induce gliadin-specific T cell tolerance in patients, preventing biopsy-confirmed small bowel pathology in celiac disease [77]. Following the positive outcome from the celiac disease clinical trial using TAK-101/TIMP-GLIA [77,88], a phase I clinical trial for the treatment of peanut allergy is presently ongoing using PLGA particles possessing similar overall physical/chemical characteristics [76]. The difference being that CNP-201 contains a purified peanut extract drug substance, as compared to the purified gliadin protein drug substance contained within TAK-101/TIMP-GLIA.

The functional mechanism of action for antigen-specific PLGA nanoparticle tolerance therapy across Th1 cell-, Th17 cell-, and Th2 cell-mediated disease models [69,77,84,87–89] is a treatment-associated increase in both antigen-specific iTregs, IL-10⁺ CD4⁺ Tr1 cells, and an increase in the expression of regulatory molecules [84,88]. Therefore, allergen-specific CD4⁺ T cells are postulated to induce expression of the same regulatory phenotype following treatment in allergy models. For example, the expression of the inhibitory molecule, CTLA-4, is induced following antigen-associated PLGA nanoparticle infusion [87]. CTLA-4 binds to CD80 (B7–1) and CD86 (B7–2). The regulatory function of CTLA-4 has been associated with CTLA-4-induced transcytosis of CD80/CD86 from APCs [90], thus decreasing the ability of these APCs to activate CD4⁺ T cells due to a decreased level of costimulatory signals. Both CD4⁺ T cells and B cells require two signals to become fully activated. First, the T cell and B cell receptors must bind their cognate antigens, either specific peptide presented in the context of MHC II or whole antigen, respectively. Second, CD28 on the surface of CD4⁺ T cells binds to CD80/CD86 on the surface of APCs (including antigen-specific B cells), and CD40 ligand (CD40L) on the surface of the CD4⁺ T cell binds to CD40 expressed by the B cell. Consequently, the T cell and B cell become activated, proliferate, and differentiate. The activation status of the CD4⁺ T cells and B cells prior to this interaction dictate the resulting outcome. For example, CD40L is expressed on the surface of previously activated T cells, but not naïve CD4⁺ T cells. B cells also differentially express CD86 and CD80 over time, with CD86 indicating early activation, while CD80 is expressed only following a decline in CD86 expression [91,92]. Similarly, CD28 is constitutively expressed by naïve CD4⁺ T cells while CTLA-4 is induced following activation while also expressed by Tregs. While CD28 and CTLA-4 both interact with CD80 and CD86, each of these proteins bind with differing affinities with CD80 having higher affinity for CTLA-4, as compared to CD28 [93,94]. The functional consequence of the interaction between CD28/CTLA-4 and CD80/CD86 on activation of CD4⁺ T cells is profound and context dependent. For example, T cells from CD28-deficient mice lack expression of CXCR5 and have a reduced ability to differentiate to Tfh cells despite the near-total absence of Treg cells [95]. Additionally, total loss of CTLA-4 expression has profound effects on humoral immunity as effector T cells produce large amounts of IL-4 leading to hyper-IgE production [96]. However, CD86 signaling internally within a B cell has been shown to increase both cell survival and increase the rate of mature Ig transcript production, resulting in an increased level of secreted antibody [97]. In contrast, the cross-linking of CD80 on the surface of a B cell both decreases the level of Ig secreted and induces apoptosis [98]. Therefore, the expression of CTLA-4 on the surface of the Treg may serve as a mechanism to further regulate the progression of the B cell response.

Additional regulatory molecules induced following antigen-specific PLGA nanoparticle treatment are PD-1/PD-L1 and IL-10, with both pathways down-regulating effector immune cell responses while promoting Treg development. PD-1 and its ligand PD-L1 are members of the B7 molecule superfamily, and PD-1/PD-L1 interaction is important for the regulation of a number of immune pathways [99]. PD-1 and PD-L1 are upregulated following activation of B cells, T cells and myeloid cells and, notably, are highly upregulated on both Tfh cells and Tfreg cells, suggesting a functional role in regulation of these cells [100]. In the context of allergy, PD-1 has been implicated in the control of humoral immunity. The interaction of PD-1 with PD-L1 induces increased levels of FoxP3 expression [101]. Defining the specific role of IL-10 on humoral responses is complicated by the ability of IL-10 to modulate the activity of T cells, B cells, and APC populations. For example, IL-10⁺ regulatory B cells have been shown to be required for the development of Tregs during an ongoing immune response [102]. While the stimulation of germinal center B cells with IL-10 upregulates Bcl-2 and prevents apoptosis [103]. IL-10 is reported to be expressed by plasma cell within the bone marrow and have regulatory function to decrease myeloid cells [104], which are also a source of plasma cell survival stimuli, i.e., IL-6 and TNF super family 13 (TNFSF13/APRIL) [105,106]. Therefore, IL-10 has a complex role in humoral immunity, with functions described for both decreasing and increasing antibody responses.

Nanoemulsions:

Nanoemulsion techniques for submicron carrier formation have also been used as treatments for allergic diseases. Intranasally delivered nanoemulsions induce an antigen-specific adjuvant effect, skewing the immune system away from Th2 cell responses and towards Treg and Th1 cell responses [107,108]. Recent mechanistic studies showed humoral changes contribute to this protection. In a casein/alum-induced murine milk allergy model, administration of four 350–400 nm intranasal nanoemulsion doses containing 20 μg of casein over a 16-week time span to sensitized mice led to a 20-fold increase in cow’s milk-specific IgG2a titers and decreased mast cell frequency in the small intestine. However, cow’s milk-specific IgE titers were still elevated following treatment [70]. These results suggest cow’s milk-specific IgE titers are not predictive of allergic reactivity, and IgG2a may play a role in providing protection by allergen blocking mechanisms or suppression of mast cell activation through engagement of the inhibitory Fc receptor, FcγRIIb. The importance of this humoral change was supported by the finding that serum from nanoemulsion-treated mice transferred to naïve mice prevented oral casein challenge-induced mast cell degranulation and hypothermia seen in intranasal casein-treated mice. Suppression of Th2 cell cytokines in the mesenteric lymph nodes was maintained for at least 16 weeks after the final nanoemulsion dose, indicating sustained unresponsiveness despite stopping treatment. Additionally, intranasal casein-treated mice increased IL-10 secretion in the absence of Th2 cell cytokine suppression, suggesting Tregs and/or Tr1 cells are not fully functional or sufficient at providing protection without Th2 cell suppression. Using the same nanoemulsion design in a murine model of multiple food allergens (egg and peanut) showed that an intranasal nanoemulsion delivering both food allergens provided protection against egg and peanut oral challenge allergic responses with 3 doses containing 20 μg of egg protein and 20 μg peanut over a 12-week time span [71]. Not only did this study prove therapeutic efficacy of delivering multiple food allergens in a multiple food allergen model, but also revealed that delivering peanut allergen alone in the nanoemulsion reduced allergic response to egg allergen through a “bystander” effect and maintained unresponsiveness for at least 8 weeks. This effect acted through the Th1-derived cytokine IFN-γ that suppressed alarmin expression of IL-25, IL-33 and TSLP and did not induce IL-17 and IL-10 found with the allergen-specific treatment. These results could be mediated by ILC2 cells since their frequencies were reduced by the bystander effect. While this type of immune response skewing may be efficacious in experimental mouse models, the clinical translation of such overall immune system response skewing may increase the risk of Th1 cell mediated autoimmune responses.

An allergen agnostic treatment using polymeric nanomicelles has shown efficacy in a murine model of peanut allergy by delivering the short chained fatty acid butyrate as an immunoregulatory molecule [72]. This approach targeted the lower gastrointestinal tract as opposed to orally administered butyrate salt, which gets absorbed by the stomach, to better induce a therapeutic effect at the site of pathology. The butyrate-conjugated co-polymer design allows for targeted release based on the charge of the polymer, with the neutral charge releasing in the ileum and the negative charge releasing in the caecum. In a cholera toxin peanut allergy model following vancomycin-induced dysbiosis, the nanomicelles were intragastrically delivered at 400 mg/kg twice daily for 2 weeks, which led to reduced anaphylaxis-related hypothermia, mast cell degranulation, and serum peanut-specific IgE and IgG1 titers. This treatment did not affect Treg populations but led to suppression of myeloid cell activation as indicated by downregulation of MHC II and CD86. Butyrate is also known to inhibit mast cell activation and could be another potential mechanism of action of the nanomicelles. These results agree with the findings in the previous nanoemulsion treatment approach [71] that allergen-agnostic approaches do not induce Tregs, but exert effects on other allergy effector cells, leading to therapeutic outcomes. These nanomicelles were also effective at reducing disease severity in a T cell transfer model of colitis, repairing epithelial barrier function in dextran sodium sulfate-induced epithelial barrier dysfunction, and inducing regrowth of Clostridia bacteria in antibiotic-induced dysbiosis [72].

Lipid Nanoparticles:

Lipid nanoparticles have been commonly used in drug delivery applications due to their ability to deliver both hydrophilic and hydrophobic molecules. Like polymeric nanoparticles, lipid nanoparticles are biodegradable, provide controlled release, and prolong circulation of cargo. Their properties can be modulated by changing lipid composition [109]. In recent years, surface studded liposomes targeting specific pathways have revealed new mechanistic insight into how they function and into how tolerance is induced. For example, 120 nm liposomes displaying peanut antigens and CD22 ligand reduced allergen-specific IgE and anaphylaxis-associated hypothermia after peanut extract/cholera toxin sensitization with two intravenous 300 μmol/mL or 600 μmol/mL doses 14 days apart. This unresponsiveness was sustained for at least 3 months after the first challenge [73]. Including CD22 ligand in combination with peanut antigens targets peanut-specific memory B cells by engaging the inhibitory CD22 receptor and induces apoptosis, preventing future humoral responses. Because the allergen-specific liposomes without CD22 ligand alone do not reduce peanut specific IgE or IgG, this finding demonstrates that CD22 targeting is required to regulate allergen-specific humoral changes and induce tolerogenic outcomes when using this lipid nanoparticle formulation. Similarly, intravenously delivered liposomes displaying allergens and ligands for mast cell inhibitory receptors inhibited IgE-mediated anaphylaxis by suppressing mast cell activation [74]. Specifically, liposomes targeting Siglec-8, a receptor with immunoreceptor tyrosine-based inhibitory motifs (ITIMs) expressed on mast cells, by displaying Siglec-8 ligand and antigen, suppressed mast cell activation through this Siglec-8-dependent pathway. This study revealed that both antigen and Siglec-8 ligand must be on the same liposomal nanoparticle in order to initiate the Siglec-8 signaling cascade since liposomes delivering only Siglec-8 or antigen alone did not confer protection. Mechanistically, this finding could be due to the IgE-FcεRI complex on mast cells needing to be in close proximity with its Siglec-8 receptor in order for phosphorylation of the ITIM to occur and induce inhibitory signaling. These B cell and mast cell targeting liposomes highlight a major advantage of a nanoparticle therapeutics, i.e. they can be engineered at a size scale that exerts specific pathway targeted effects with precision at a physiologically relevant conformations.

In recent years, lipid nanoparticles have been developed that deliver nucleic acids more efficiently. Delivering nucleic acids instead of allergen potentially increases the safety profile of the therapeutic by reducing treatment-related adverse events usually seen in AIT since no allergen protein is being delivered but rather nucleic acids that do not have the epitopes that are recognized by allergen-specific IgE. Nucleic acid delivery is limited by in vivo instability due to nucleases, by loading efficiency in nanoparticle systems based on nucleic acid length, and by identifying specific targets, i.e., in food allergies, the exact epitope driving allergic responses is not always known. However, it is expected mRNA-loaded nanoparticles would function by similar mechanisms as peptide- and protein-loaded nanoparticles, since the same cell types would uptake the nanoparticles and mRNA would be translated into peptide or protein and then presumably processed in the same way as peptide or protein delivered by protein-loaded nanoparticles. In a murine model of peanut allergy, liver-targeting, 148.7 nm, −4.33mV, lipid nanoparticles surface decorated with mannose ligand delivering mRNA for MHC-II binding epitopes of the dominant peanut allergen, Arachis hypogaea protein 2 (Ara h2), showed efficacy at reducing allergic symptoms as well as inducing IL-10 producing Tregs [75]. These particles conferred therapeutic effect with two 1.25 mg/kg intravenous doses 1 week apart in a cholera toxin-peanut extract induced sensitization. This mRNA approach could allow for delivery of multiple epitopes within the same nanoparticle.

Conclusions:

Nanoparticle-based therapies have been under development for several decades with efforts focused on designing nanoparticle carrier systems with enhanced pharmacokinetics, improving the delivery of therapeutic agents, and reducing toxic side-effects. Additionally, the biocompatibility, tunable physiochemical properties, ease of functionalization, and ability to overcome biological barriers of biodegradable polymers make nanoparticle-based therapies an attractive next generation therapeutic. The surface of these nanoparticles can also be functionalized such that specific cell populations may be targeted. While the specific cellular mechanism(s) of action for each nanoparticle-based therapy may differ, future mechanistic studies will help to instruct next generation nanoparticle formulations (Table 2). Emerging evidence suggests that defects in Treg function may contribute to the development and progression of food allergies. Studies have identified reduced suppressive capacity and diminished stability of Tregs in individuals with food allergies [33,34]. These alterations may lead to a breakdown of immune tolerance, allowing for the activation and proliferation of effector T cells specific to food antigens, leading to an exaggerated immune response to harmless food antigens. The development of targeted antigen-specific therapeutic strategies that suppress Th2 immune responses and enhance Treg function hold promise for effective, safe and durable management of food allergy. Several such approaches have shown promise in pre-clinical mouse models, with several currently in clinical trials.

Table 2.

Unknowns in the field of nanoparticles as a food allergy therapy

1. Mechanisms of nanoparticle mediated therapy are being researched in murine models of food allergy but are not fully understood nor uniform between different nanoparticle types.

2. Safety and efficacy of allergen-loaded nanoparticles in human clinical studies have yet to be tested.

Abbreviations:

AIT

Allergen-specific immunotherapy

AK

Arginine kinase

APCs

Antigen presenting cells

Ara h2

Arachis hypogaea protein 2

BLG

β-lactoglobulin

CTLA-4

cytotoxic T lymphocyte-associated protein 4

DCs

Dendritic cells

OIT

Oral immunotherapy

EPIT

Epicutaneous immunotherapy

FcɛR1

high-affinity IgE receptor

FoxP3

Forkhead box P3

Ig

immunoglobulin

ILCs

Innate lymphoid cells

IL

interleukin

ITIMs

Immunoreceptor tyrosine-based inhibitory motifs

LAG-3

lymphocyte-activation gene 3

MHC II

Major histocompatibility complex class II

PD-1

programmed cell death protein 1

PLGA

Poly(lactic-co-glycolic acid)

PVA

Polyvinyl alcohol

PEMA

Poly(ethylene-alt-maleic anhydride)

SLIT

Sublingual immunotherapy

SCIT

Subcutaneous immunotherapy

Th cells

T helper cells

Th2

T helper cell type 2

Th1

T helper cell type 1

Tfh

T follicular helper cell

Tregs

T regulatory cells

TSLP

thymic stromal lymphopoietin

XLAAD

X-linked autoimmunity-allergic dysregulation

OVA

Ovalbumin