There is a delicate balance between immune tolerance and responsiveness against foreign assault. If the balanceis shifted towardstolerance it may underpin the development of pathological conditions, such as cancer. However, if the immune system is overly responsive, this may induce autoimmune diseases and allergic disorders (1,2). Hyper responsiveness of the immune system is responsible for different allergic conditions in atopic individuals (3). Studies have estimated that >25% of the population in developed countries suffers from immunoglobulin (IgE)-mediated allergies or ‘type I hypersensitivity’ (4–6). Allergen-specific immunotherapy (AIT), also known as ‘allergy vaccination’ or ‘desensitization’, is a treatment that fine-tunes the defense system of the body to become tolerant to a specific allergen over a period of time (7,8). AIT is accompanied by several potential drawbacks, such as local and systemic immune reactions during AIT administration, and variable patient outcome (9,10). Despite the risk and differential response among individuals, AIT is still the most effective approach and the only therapeutic approach that is specific for the treatment of allergy. AIT reduces the activation and proliferation of lymphocytes in response to allergenic stimulus and further enhances the immune tolerance mechanisms towards specific allergens (11). Principally, during AIT, the allergen is administered to the host via different routes at increasing concentrations to achieve an effective immunotherapy (12). Theprocessis divided into twophases: The ‘build up phase'and the ‘maintenance phase’ (13).

During the first phase, or ‘build up phase’ of immunotherapy, an increasing dose of the therapeutic formulation is administered to the host 2 or 3 times in a week, which enhances the allergen tolerance over time (14). The length of this phase varies based on the frequency of injections and effectiveness of the therapeutic formulation but generally ranges between 3 and 6 months (14,15). After achieving an effective dose in the ‘buildup phase’, the second phase of AIT is started as a ‘maintenance phase’ (15). The gap between therapeutic injections throughout the maintenance phase varies and may range from 2–4 weeks to 2–5 years (15,16). This desensitization process increases the threshold dose of the allergen to cause allergic reactivity (13). Several studies have provided evidence that administration of a suitable allergen for immunotherapy not only provides protection against its own allergenicity but also reduces the probability of developing sensitization against other allergens (17,18). The first specific immunotherapy for grass pollen-induced hay fever was introduced in 1903 (19). However, over time, the treatment options with different routes for AIT expanded the scope of immunotherapy to other allergic diseases (20–24). The identification of IgE and a blocking antibody of IgE, IgG4, provided a leap forward in the field of AIT (25,26). With further advances in technologies in later years, such as the synthesis of chemically modified and genetically engineered allergens with low allergenic activity and their use for AIT, the scope of modifying the allergens for an improved clinical outcome broadened (27,28). In addition, novel biomarkers have been identified that could be useful for monitoring the effectiveness and predicting the safety of immunotherapy (29). The practices of allergen immunotherapy have been knowingly or unknowingly used for several decades. Fig. 1 provides a roadmap of findings associated with AIT (19–30). The present review attempts to provide a comprehensive overview of the history and diverse clinical applications of AIT, and to explore developments in the scientific understanding of therapy along with future perspectives.

Allergens are a complex mixture of allergenic and non-allergenic ingredients comprising single or multitudinous combinations of proteins, carbohydrates, lipids and glycoproteins (31). The allergenic ingredients that are responsible for induction of allergy could potentially also be used for the diagnosis and specific immunotherapy of the same allergy (32). Usually, the therapeutic formulations are directly prepared from a natural source of allergen, which contains allergenic as well as non-allergenic components (31,32). The concentration of an allergen for inducing effective immunotherapy depends on several bio-variable factors, such as the ratio of allergic and non-allergic ingredients, their quantity, processes used for their isolation and purification, and genetic makeup of the affected atopic individual (33). Crude allergenic extract is extensively used for immunotherapy; however, four major problems are often witnessed during the course of this method: i) Allergen extracts comprise a variety of allergenic as well as non-allergenic proteins, other macromolecules and toxic ingredients, which is often difficult to standardize involving high batch-to-batch variability; ii) development of specificities against newer proteins; iii) unpredictable clinical response due to systemic administration of intact allergens through extracts; and iv) effective therapeutic doses are often difficult to achieve due to a lack of standardized extracts (33–35). Therefore, the standardization of allergen extracts from their natural source is necessary to diminish their allergenic potential and to ensure their consistent composition and potency for AIT (36). At present, various injectable and non-injectable, Food and Drug Administration (FDA)-approved allergen extract-based therapeutic formulations are used for the diagnosis and treatment of different allergic diseases (37–39). However, the majority of the FDA-approved allergen extract-based therapeutic formulations are non-injectable. For example, to treat allergic rhinitis and conjunctivitis, the FDA-approved GRASTEK (timothy grass pollen allergen extract), ODACTRA (house dust mites allergen extract), RAGWITEK (short ragweed pollen allergen extract) and ORALAIR (sweet vernal, orchard, perennial rye, Timothy and Kentucky bluegrass mixed pollens allergen extract) are available as tablets for sublingual AIT (37,40–42). Similarly, PALFORZIA (peanut allergen powder) is also an FDA-approved allergen extract-based therapeutic formulation that is used for the treatment of peanut allergy through oral immunotherapy (OIT) (38,43). In addition, numerous other injectable and non-injectable allergen extract-based therapeutic formulations are being investigated in different phases of clinical trials (39,44,45). Table I provides an overview of allergen extracts approved by the FDA or undergoing clinical trials.

At present, novel ways are being developed to introduce chemical modifications in the allergenic extracts intending to lower their allergenic potential without affecting the immunogenicity, and such modified extracts are termed as ‘allergoids’ (27,46,47). To enhance the efficacy of the immunotherapeutic approaches and decrease the allergenic properties of a given protein, different chemical, structural and recombinational modifications can be introduced in the allergen (34). The generation of hypoallergenic hybrid molecules through conjugation of allergens to adjuvant substances activating innate immune cells, such as CpG oligonucleotides, carbohydrate-based particles, or nanosized therapeutic formulations are examples of these modifications (48–50). These alterations are primarily intended to modify the IgE-specific epitopes present on allergens, while keeping the T cell epitopes intact (51). Methods of chemical modifications to prepare hypo allergens are advantageous over others as they can be applied on different homogenous as well as heterogeneous types of allergens. For example, coupling of allergens with polyethylene glycol, glutaraldehyde and formaldehyde has been illustrated to modify the IgE epitopes of allergens (34,51). Similarly, treatment of allergens with maleic acid anhydride generates recognition sites for different scavenger receptors in allergens, thereby facilitating their intake by phagocytic cells, and immunotherapy with these hypo allergens has been observed to induce the type 1 helper cell (Th1) dominated immune response (52,53). Similarly, conjugation of allergens with synthetic oligo-deoxy nucleotides carrying immune-stimulatory CpG sequences from bacterial DNA can mask IgE-specific epitopes on allergens and could potentially block the cross-linking between allergens and IgE bound to high affinity IgE receptor (FcεRI) present on the surface of mast cells and basophils (54). In order to further refine this strategy, further investigations that compare the relative efficiency of the chemical modifications and determine their potential synergistic or additive effects are required.

Recombinant DNA technology has also been used for targeted alteration in allergenic proteins by means of mutations, deletions, fusion, site-directed mutagenesis and hybridization (32). Recombination of the genes of allergens requires knowledge of their sequences and positions of amino acids, as well as their three-dimensional conformation, which is helpful for targeted modification (34,55). Using this approach, hypo allergens for the timothy grass Phil p 5b and American Cockroach Per a 1 allergen have been successfully prepared by deleting the IgE epitopes present in the corresponding gene segments, and the consequent hypo allergens were observed to have reduced IgE binding properties, reduced histamine-releasing activity and reduced skin reactivity (56,57). After modification, allergens may exhibit low allergenicity but also carry the risk of generating new epitopes that may induce allergic reactions (48). Therefore, it is important to subject the newly synthesized hypo allergens to pertinent in vitro and in vivo evaluation tests before approving them for therapeutic applications.

Allergy is fundamentally an undesirable hyperactive immune response to allergens, which occurs due to a breach of peripheral tolerance and dysregulation of immune homeostasis mediated by cellular and molecular factors, such as TLR4 or TLR8, regulatory T cells (Tregs), T cell, immunoglobulin and mucin and allergen-specific MHC class II tetramer⁺ cells (58,59). Forkhead box P3 (FoxP3)-positive Tregs are pivotal for generating tolerance against self-antigens and harmless non-self-antigens (60,61). Usually, Tregs present at the mucosal surfaces suppress the immune cells involved in the mediation of allergic responses, such as type 2 CD4⁺ helper cells, mast cells and eosinophils (62,63). Distinct approaches have been used in various AIT studies; however, there is a profound overlap in the mechanism underlying these AITs and their allergen-specific tolerogenic features (64,65). The major differences among various approaches primarily involve the role of antigen presenting cells (APCs) associated with differentroutes of immunotherapy, memory cell or Treg responses, characteristic immunoglobulins produced, and interaction with other immune cell types present at the interface niche, where the primary encounter of the tolerogenic protein occurs with the host (12,66,67). Notably, the APC phenotype present at the host-environment interface servesan important role in peripheral insensitivity or immunogenicity to innocuous antigens (68,69). For example, dendritic cells (DCs) are specialized antigen-presenting cells, which initiate and sustain allergic inflammation, or support tolerance induction (70,71). After being triggered by an antigen, immature DCs polarize into either dendritic cells-1, dendritic cells-2 (DC2s), dendritic cells-17 or regulatory dendritic cells (DCregs), which induce the differentiation of naïve T cells into Th1, type 2 helper (Th2) or T helper 17 cell (Th17), or Tregs, respectively (29,71,72). Compared with immature DCs, DCregs or tolerogenic DCs represent an intermediate stage of DC maturation characterized by higher expression levels of class II major histocompatibility complex (MHC) and co-stimulatory molecules, but often lack the capacity of proinflammatory cytokine secretion (73,74). The DCs involved in AIT are primarily myeloid DCs (mDCs) and Langerhans cells (LCs), which are also characterized by expression of FcεRI on their surface (75,76). mDCs secrete IL-12, which tilts the Th1/Th2 balance towards Th1 responses, whereas LCs promote the development of T helper 3 regulatory cells via production of IL-10 and TGF-β, thereby attenuating the Th2 immune responses (71,75). The induction of allergen-specific Tregs is the central factor in all types of AITs; however, how the allergen-specific effector T cells or naïve T cells transform into allergen-specific Tregs is not well understood. It would appear that the myriad of signals present in the microenvironment of immature DCs after the phagocytosis of allergens orchestrates the development of the Th2-mediated allergic response or tolerogenic response against allergens spearheaded by Tregs (76,77). It has been demonstrated in several models that coincident exposure of pathogens or endotoxin with allergen may lead to onset of IgE-mediated allergic responses (78,79). However, in the absence of pathogenic signals during AIT, the immature DC sunder go tolerogenic interaction with T cells of the lymph node (80). This promotes the development of IL-10, TGF-β and IL-35-secreting Tregs, thereby inducing allergen-specific peripheral tolerance (81–83).

These suppressive cytokines (IL-10 and TGF-β) are known to inhibit the differentiation, proliferation and activation of effector T cells, and further bring about desensitization of mast cells and basophils (84). IL-10 acts by decreasing the production of allergen-specific IgE, while increasing the levels of immunoglobulin G4 (IgG4) and immunoglobulin G2a (IgG2a) secretion from B cells (85). In addition, TGF-β is also involved in the induction of allergen-specific tolerance during AIT (86). Tregs are the major source of TGF-β, which affects T cell proliferation and differentiation, and inhibits Th2 differentiation by suppressing GATA binding protein 3 (GATA-3) expression and IL-4-mediatedSTAT6 activity (87–89).

Apart from the suppressive Tregs and DCregs, a population of IL-10-secreting suppressor B cells has also been identified, and this is known as regulatory B cells (Bregs) (90,91). The primary function of Bregs is to support immunological tolerance and inhibit unwanted inflammation (92). IL-10-secreting Bregs serve an important role in the tolerance induction during AITs (93). IL-10 is a key suppressive cytokine associated with Bregs; however, TGF-β and IL-35 have also been identified as Breg-associated suppressor molecules (94,95). Different subsets of Bregs have been described in humans and a defective development and function of Bregs may result in various chronic inflammatory diseases, such as collagen-induced arthritis and chronic hepatitis B virus infection (96,97). Although IL-10 secretion is common to all Bregs, they are further grouped into different subsets based on their differential functions (98). The immature/transitional Bregs (CD19⁺CD24^hiCD38^hi) suppress effector T cells but enhance Treg function (99). Similarly, another sub-population of Bregs, known as memory B Cells/B10 Bregs (CD19⁺CD24^hiCD27⁺), enhance and stabilize the expression of FoxP3 in Tregs (100,101). Another subset of Bregs (CD25^highCD71^highCD73^low) prevents peripheral tolerance by producing IL-10 and blocking antibody IgG4 (93,102). Notably, it has been reported that the relative percentage of CD19⁺ CD24^hiCD27⁺ Bregs was decreased in patients with allergic rhinitis, whereas an increase in the percentage of CD19⁺CD24^hiCD38^hi Bregs was observed in comparison with healthy individuals; however, the significance of this finding is unclear (103). It has been noted that Bregs serve a critical role in effective AIT. After AIT, the percentage of IL-10 and IgG4-secreting Bregs increases, which suppresses the allergen-specific CD4⁺ T-cell proliferation and further ameliorates the allergic airway inflammation via FoxP3-positive T regulatory cells (93,104). In another study conducted on bee venom antigen allergic patients subjected to AIT, an enhanced percentage of IL-10 and IgG4-secreting CD25^hiCD71^hiCD73^low Bregs, which potently suppress allergen-specific CD4⁺ T-cell proliferation and produce increased amounts of IgG4, was found (93). Notably, a 10-100-fold increase in serum allergen-specific IgG4 isotype has been observed for AITs (85). IgG4 functions as a blocking antibody for IgE and considerably reduces the binding of IgE to its receptor present on the surface of mast cells and basophils (105). This process prevents mast cell degranulation, which in turn downregulates the activity of eosinophils and neutrophils (106). IgG4 antibodies also inhibit the proliferative response of T-cell clones by blocking IgE-facilitated allergen binding to B cells and thereby inhibiting the presentation of allergenic peptides by B-cells to allergen-specific T-cell clones (93). Additionally, an increase in IgG2 a in the serum shifts the Th1/Th2 immune response towards a Th1-dominated immune response (107). Despite early generation of Tregs following AIT, it may still take years to effectuate a marked reduction in IgE levels in the allergic individuals (108). An analysis of the mechanisms of AIT has been summarized in Fig. 2, and a comparison of various AITs is presented in Table II.

The most important factor, which contributes to the duration of AIT, is the route of administration and this has a marked influence on the clinical outcome of immunotherapy (12). There a large variations in the immune niche present at various external tissue interfaces associated with different AITS, which serve a major role in fine orchestration of immune responses (12,109,110). The routes of administration of AIT can be categorized into subcutaneous immunotherapy (SCIT), sublingual immunotherapy (SLIT), OIT, intralymphatic immunotherapy (ILIT) and epicutaneous immunotherapy (EPIT).

Since the fundamental process of immunotherapy involves challenging the sensitized patients with increasing doses of allergen, there is always an acute possibility of undesirable minor to severe allergic reactions. Furthermore, during the ‘build up’ or ‘escalation’ phase of immunotherapy, local and systemic reactions are often witnessed with increasing doses, which impedes the procedural efforts to achieve a therapeutically active ‘maintenance’ dose (15). Furthermore, due to huge variations in the sensitivity of different patients to an allergen, the therapeutic formulation applicable for one atopic individual may not be promising for others (10). Another confounding factor is the variation in the composition of allergenic extracts available for immunotherapy from different manufacturers, which may arise due to differences in allergen sources and allergen extract preparation protocols (171). Allergen extracts prepared from divergent natural sources may get contaminated with pathogens and allergens from other sources, which yields undesirable immunogenicity and even new IgE-mediated allergies (46). One of the causes underlying the limited success and variable outcome of immunotherapeutic approaches so far is the absence of standardized procedures and regulatory guidelines for preparation of allergen extracts and their characterization (10,172). Another major bottleneck hindering the progress of AIT is the lack of appropriate biomarkers that can predict the efficacy of AIT (29).

Another growing concern is the problem of ‘polysensitization’, which is a sensitivity of atopic individuals to two or more allergens, and this condition is referred to as ‘polyallergy’ after clinical confirmation (185). According to estimates, 60–80% of allergic patients are polysensitized (186). An increasing prevalence of polyallergy has been documented with age, which necessitates the development of immunotherapeutic approaches that can take care of more than one allergen simultaneously (186,187). However, an additive preparation of mixture of allergens for simultaneous AIT may not yield the desirable outcome, as one allergen may affect the stability and optimal dose of the other allergen, thus affecting its immunotherapeutic potential, efficacy and even safety (188–190). In this regard, the European Medicines Agency has suggested that AIT should not be performed with a mixture of two non-homologous allergens, and should be performed separately for seasonal or perineal allergens (10). This creates the problem and annoyance of enduring separate immunotherapeutic procedures by patients for addressing multiple allergen sensitivities on an individual basis (190). The polyallergic condition in patients may arise due to ‘cross-reactivity’ or ‘co-sensitization’ (186). Cross-reactivity is defined as IgE reactivity against structurally related proteins when the sequence homology is often >70%, whereas co-sensitization may involve multiple IgE sensitizations against structurally unrelated allergen groups (191). It is essential to understand the nature of polyallergy with respect to ‘cross-reactivity’ or ‘co-sensitization’ to design a safe and effective AIT (192). With the advancement in science and technology, it is now possible to isolate the pure allergic components from their natural source for refined diagnosis and treatment of allergies. Component-resolved diagnosis (CRD) utilizes purified native or recombinant allergens to detect IgE sensitivity against individual allergen molecules and has assumed increasing importance in clinical investigation of IgE-mediated allergies (193). The CRD technique quantifies serum specific IgE against individual allergenic proteins or even allergenic peptides present in natural sources, rather than quantifying IgE against the whole natural extract (194). At present, CRD diagnosis is used in laboratory practices as single plex and multiplex arrays and offers a promising technology that could replace conventional serum specific IgE assays in the near future (195). One of the major advantages of CRD is that it can discriminate true allergens from the cross-reactive allergen molecules and polyallergy of other related allergens (196). However, CRD analysis utilizes intact proteins or random peptides in its present form, which makes the data interpretation complex and ambiguous (192,196). A more refined CRD approach could entail the use of individual IgE epitope-based recombinant fragments present in a protein, rather than using the whole allergenic protein components (192). In silico analysis in conjunction with wet lab validation allows determination of specific IgE epitopes present in an allergen, which can be further employed for predicting epitope specific IgE reactivity of patient serum (197). The present review describes a strategy for developing allergy arrays with potential application for AIT in patients with polyallergy (Fig. 3).

The strategy offers a simple and robust tool with a high resolution for predicting IgE cross-reactivity or co-sensitization from single or multiple allergenic sources. After a thorough characterization of the IgE sensitivity profile of a patient, the same IgE epitope-based recombinant fragments can also be used for generating hypoallergens intended for use in AIT (198,199). The hypoallergen could be prepared by modifying the IgE specific epitopes of the particular allergen either by coupling them with chemical modifiers or by altering the coding sequence of the allergy-responsive component of the allergen using recombinant DNA techniques (46,200,201). A combination of these hypoallergenic epitope-based proteins may be employed for AIT through a single dosing regimen plan (202). However, before the onset of AIT, it should be ensured by a skin prick test that the serum of the patient shows IgE reactivity towards the allergens but not the hypoallergens (202).

There is a need for devising strategies aiming at improved predictability of AIT, minimization of side effects, annoyance of injection, irritability, fatal outcomes and a shorter immunotherapeutic duration along with sustenance of life-long tolerance for the allergen. Several combinatorial therapies, which involve administration of allergen extracts with immunomodulatory or suppressive cytokines, such as TGF-β, IL-35 and IL-10, have yielded encouraging results; however, these approaches may markedly escalate the cost of immunotherapy (203,204). Different endogenous specialized proresolving lipid mediators (SPMs) have also shown promise as therapeutic agents in the resolution of allergic inflammation. Results from several experimental systems indicate that SPMs, including lipoxins, resolvins, maresins and protectins, are multi-pronged and potent regulators of inflammation and stimulate resolution (205,206). For example, a combination of resolvin D1 (RvD1) and 17-hydroxydocosahexaenoic acid has been demonstrated to inhibit IgE production by human B cells and it also suppresses the differentiation of naïve B cells into IgE-secreting cells by specifically blocking epsilon germline transcript (207). Furthermore, other studies have also investigated the roles of SPMs in murine models of allergic airway inflammation and have revealed their protective role in allergic asthma (208–210). RvD1 is also known to reduce the allergic airway inflammation by targeting eosinophils and proinflammatory mediators involved in the Th2 signaling pathway, while resolvin E1regulates the development of Th17 cells and IL-23 production (205). Similarly, exogenous administration of maresin1 (MaR1) during the allergen challenge phase attenuates allergen-triggered inflammation by decreasing the multiple allergy-associated parameters, such as numbers of eosinophils, allergen-specific IgE levels and type 2 cytokines in bronchoalveolar lavage fluid (BALF), and increasing TGF-β levels (211). The MaR1-mediated increase in BALF TGF-β triggers Tregs to limit type 2 innate lymphoid cell activation, and thus, promotes resolution of lung inflammation. In addition, MaR1 promotes lung catabasis for allergic asthma by suppressing ILC2-derived IL-5 and IL-13, while stimulating the expression of amphiregulin (211). Furthermore, amphiregulin itself contributes to a constitutive, low-level release of bio-active TGF-β within tissues, leading to continuous tissue regeneration and to an immunosuppressive environment, which may keep inflammation-prone tissues in the homeostatic state (212). Considering the multi-pronged beneficial actions of SPMs, they are important potential candidates for combinatorial AITs.

Furthermore, as aforementioned, the lack of appropriate biomarkers indicating successful progression of AIT is a major bottleneck affecting the clinical outcome of allergy immunotherapy. Clinical investigations examining the expression levels and biosynthesis of SPMs in relation to efficacious AIT may help to identify prospective biomarkers. In a model of allergic lung inflammation, MaR1 production declined upon allergen challenge but increased with resolution of allergic inflammation (211). This finding suggests that levels of MaR1 in tissues before and after allergen-specific immunotherapy may be tested as a biomarker of successful immunotherapy.

The knowledge gathered in the past decades has helped in developing an improved understanding of the mutual interaction between immune cell types presents in diverse immunological niches, thereby propelling the evolution of different AIT routes of administration and improved therapeutic formulations. As a noteworthy breakthrough, the over-the-counter sale of certain AIT formulations is also now possible, the self-administration of which does not require any special hospital supervision. However, there is still much to be done to address the issues of standardizing AIT formulations, the risk of frequent adverse reactions, the maintenance of tolerance to allergens, the reduction in the duration of AIT, and other concerns, such as polyallergy. Developing immune tolerance against allergens is the primary aim of AIT but the current understanding of the precise mechanism underlying the induction of allergen-specific Tregs is still in its infancy. An improved scientific understanding of key events guiding antigen-specific tolerance would pave the way for the advent of novel non-invasive technologies targeting induction of allergen-specific Tregs for an improved prognosis of AIT and complete cure of allergies.