The use of Allergoids and Adjuvants in Allergen Immunotherapy

Allergen immunotherapy (AIT) is the unique curative approach to treat allergic diseases [1]. Since the seminal work published by Curtis in the New York Medical Record of July 13, 1901 employing “drops” of tincture or luid extracts of pollens prepared by a botanist, several strategies have been employed to induce tolerance to speci ic allergens in order to improve the quality of life of allergic patients [2]. The experimental induction of oral tolerance in laboratory rodents followed the clinical assays and began its irst steps in 1909 [3]. The further steps were to translate the controlled laboratory experiments to the real diversi ied human clinical disease management [4]. One century of laboratory and clinical experience have shown us that the choice for a tolerance-induction strategy may rely on a more dose-controlled antigen administration as provided by subcutaneous shots, or may rather explore a more cost-effective tolerogenic route, like the sublingual/oral administration, that may induce tolerance by several mechanisms along with low or high doses [5,6]. Usually, to avoid the undesired side effects of its natural reactivity, the AIT is planned with incremental doses of the same unmodi ied offending allergens that are extracted and isolated from similar sources that originated the clinical symptoms, the so-called allergen-speci ic immunotherapy (ASIT) [7]. This is in contrast with the so-called nonspeci ic desensitization that employs correlate antigens or simple microorganismlike unspeci ic adjuvants to modify the Th1/Th2 balance and immune deviation [8,9]. To produce desensitization, the initial administered dilutions must not elicit allergic reactions and, as the ASIT progresses with increased concentrations of allergens, the induced tolerance mechanisms suppress the hypersensitivity reactions, allowing the prescription of higher concentrations of the natural allergens [10]. Therefore, the main limitation of ASIT is its intrinsic allergenicity derived mainly from its conformational epitopes recognized by speci ic antibodies, while its therapeutic (immunogenic) activity derives from its hidden linear epitopes, recognized by T regulatory (Treg) cells after proper intracellular antigen-presenting cell (APC) digestion and intercellular class II major histocompatibility complex (MHC) presentation to the T cells receptors (TCR) [11]. The presentation of the linear epitopes to the Treg cell in a non-in lammatory context induces the production of regulatory cytokines Abstract


Introduction
Allergen immunotherapy (AIT) is the unique curative approach to treat allergic diseases [1]. Since the seminal work published by Curtis in the New York Medical Record of July 13, 1901 employing "drops" of tincture or luid extracts of pollens prepared by a botanist, several strategies have been employed to induce tolerance to speci ic allergens in order to improve the quality of life of allergic patients [2]. The experimental induction of oral tolerance in laboratory rodents followed the clinical assays and began its irst steps in 1909 [3]. The further steps were to translate the controlled laboratory experiments to the real diversi ied human clinical disease management [4]. One century of laboratory and clinical experience have shown us that the choice for a tolerance-induction strategy may rely on a more dose-controlled antigen administration as provided by subcutaneous shots, or may rather explore a more cost-effective tolerogenic route, like the sublingual/oral administration, that may induce tolerance by several mechanisms along with low or high doses [5,6]. Usually, to avoid the undesired side effects of its natural reactivity, the AIT is planned with incremental doses of the same unmodi ied offending allergens that are extracted and isolated from similar sources that originated the clinical symptoms, the so-called allergen-speci ic immunotherapy (ASIT) [7]. This is in contrast with the so-called nonspeci ic desensitization that employs correlate antigens or simple microorganismlike unspeci ic adjuvants to modify the Th1/Th2 balance and immune deviation [8,9]. To produce desensitization, the initial administered dilutions must not elicit allergic reactions and, as the ASIT progresses with increased concentrations of allergens, the induced tolerance mechanisms suppress the hypersensitivity reactions, allowing the prescription of higher concentrations of the natural allergens [10]. Therefore, the main limitation of ASIT is its intrinsic allergenicity derived mainly from its conformational epitopes recognized by speci ic antibodies, while its therapeutic (immunogenic) activity derives from its hidden linear epitopes, recognized by T regulatory (Treg) cells after proper intracellular antigen-presenting cell (APC) digestion and intercellular class II major histocompatibility complex (MHC) presentation to the T cells receptors (TCR) [11]. The presentation of the linear epitopes to the Treg cell in a non-in lammatory context induces the production of regulatory cytokines such as IL-10 and TGF-ß with further decrease of the B cell production of reaginic antibodies (such as the IgE), stimulation of the regulatory B cells (Breg) and increase of the production of blocking antibodies (such as the secretory IgA and IgG4) [12][13][14][15][16]. Blocking antibodies act competing with reaginic antibodies [17]. Secretory IgA acts on mucosal surfaces ensuring immune exclusion and immune modulation [18]. IgG4 promotes tolerance due to its functional bivalence and incapacity to ix complement [14,19]. However, the administration of allergens in an in lammatory context, as it occurs with the progression of the subcutaneous shots, also stimulates the production of the less pathogenic Th1 cytokines, a fact that may be bene icial for the treatment of Th2 allergic diseases but can theoretically induce an undesirable T cell reactivity [20]. In order to decrease the unpleasant collateral effects of ASIT derived from its intrinsic allergenicity the medical community conceived the production of allergen derivatives, that are generically known as allergoids [21]. An allergoid is a modi ied allergen designed to have a reduced allergenic activity, when compared with the native allergen, along with a suf icient immunogenic activity [22]. The use of allergoids was inspired in the work of Lowenstein (1909) who inactivated the toxic activity of the tetanus toxin by the use of formaldehyde, producing an immunogenic non-toxic derivative [23]. The irst claimed conception for the use of allergoids for allergic desensitization was registered by Carter, from Abbot Laboratories, in the US patent 2,019,808 (1935), where he described the techniques to produce detoxi ied pollen extracts. Since then, the clinical bene its and safety of the use of allergoids in AIT strategies have been demonstrated in several double-blind placebo controlled clinical trials [24]. The use of allergoids in an AIT tolerance-induction strategy is rationalized by the decrease in number of conformational B cell epitopes recognized by IgE reaginic antibodies (less allergenicity) and maintenance of the IgG epitopes and linear T cell epitopes (immunogenicity) [25][26][27][28]. The irst studies about allergoids demonstrated that they did not combine with cell-bound IgE (i.e. showed no "hapten-like" activity) due to their incapacity to inhibit the histamine release from leucocytes [29]. In fact, longterm clinical studies performed with speci ic subcutaneous allergoids demonstrated a low risk of serious adverse reactions [30]. The conception that the allergenic conformational epitopes may not be involved in the mechanisms for desensitization leads to the understanding that, in a tolerance induction strategy, it is not necessary to employ all the immunoreactive epitopes of a given speci ic allergen [31]. The use of one or few immunodominant T cell linear epitopes is able to produce tolerance to the original allergen and even to related allergens inducing the so-called linked epitope suppression [32]. However, the fact that the allergoid has a diminished IgE-mediated reactivity does not exclude the possibility of a hypersensitivity reaction mediated by a non-IgE mechanism such as the type IV of Gell and Coombs [33]. The great challenge to produce more convenient allergens to the clinical management of allergic diseases stimulated the scientists to develop a molecular approach to a better understanding of the in vitro and in vivo mechanisms of allergenicity and immunogenicity [34]. In order to develop a product for a tolerance-induction strategy it is convenient that the scientist and the practitioners possess objective tools to evaluate the allergenicity and the immunogenicity of the modi ied antigens, that are candidates to be used in clinical settings [35]. Several clinically accessible tools can evaluate allergenicity such as speci ic IgE dosage, the allergic skin tests, the provocation tests and the basophil degranulation tests [36,37]. The humoral and cellular immunogenicity of a given allergoid may generically be evaluated by the inal production of allergic-speci ic blocking antibodies or with help of cellular ex vivo tests, such as the dosage of Th1/ Th2/Treg cytokines, the lymphocyte proliferation assay, the leukocyte adherence inhibition test, the leukocyte migration inhibition test or the degradome assay [38][39][40][41][42][43]. Some allergoids have suf icient immunogenicity to be used alone, but some ones, specially the short ones do not have, per se, enough immunogenicity, what raises the need of the association with adjuvants, such as proteic carriers and immune stimulants to amplify the desired immune response [44]. The safety and ef icacy of allergoid immunotherapy turned it, now a day, a real tool for desensitization [45]. The lack of allergenicity of allergoids allows their use in accelerated up-dosing schemes, as well ultra-short-course boosters, shortening the immunotherapeutic process [46][47][48][49]. We can ind in the literature different ways to produce allergoids and adjuvants. In this review, we expose some of these techniques.

Extraction and purifi cation methods
The irst AIT preparations were done with help of physical methods such as freezing, thawing, boiling and dilution (in distilled water or salt solutions) of the natural sources related with the patient's symptoms, in order to "extract" the active constituents whose allergenicity was further corroborated by conjunctival and/or skin tests [50]. One of the very irst problems to be solved was the dilution of the extract, since the irst undiluted solutions originated severe reactions that prevented the continuity of the treatment [51]. Allergologists also realized that only one or few components of the allergen's extract were responsible for the symptoms, and with help of puri ication methods, they began to isolated proteins to perform the diagnosis and the speci ic immunization [52]. Soon, several extraction solutions, idealized to alter the solution's pH, and therefore the ionic liaisons between the constituents, began to be employed in the preparation of the extract [53]. Similarly, additional solutions were idealized for further dilution and conservation of the diagnostic and therapeutic preparations, such as the glycerin, a good preservative agent with the inconvenient to produce pain at the injection site at larger concentrations [54]. Besides preservation, high concentrations of glycerin have the capacity to inactive catalytic activity of allergens such as molds and insects, allowing their combination without loss of immunogenicity [55]. Initially, allergists employed only nonstandardized personalized allergen's extracts but now a day, standardized, commercially available and ready-to-use preparations, with one or multiple allergens were added to the clinical arsenal [56]. When the extraction solution also submits the allergen's extract to an acid and/or alkali treatment and concomitant dialysis in order to remove substances attached to the proteins by ionic liaisons, the process is called depigmentation [57]. Depigmentation signi icantly decreases the enzymatic activity and the allergenicity of active extracts [58]. Now a day, depigmentation is considered a preliminary step to further process of the AIT preparations [59].

Allergen modifying processes
Thermal Denaturation: Cooking is an universal, ancient and domestic way to produce allergoids [60]. Thermal processing of proteins can potentially denature some of their conformational epitopes, decreasing their allergenicity and keeping their immunogenicity by the concomitant preservation of the linear epitopes [61]. Additionally, thermal processing may render thermolabile allergenic proteins more susceptible to peptic digestion [62]. Thermal denaturation is used in the desensitization of cow's milk-allergic patients [63]. However, heat denaturation may also expose new antigens by unfolding the proteins, enhancing their allergenicity [64].

Chemical Denaturation;
In the era before the academic knowledge of the T-cell, formalin, tannic acid and other chemicals were used by allergists to denature and precipitate pollen's extracts in order to reduce their "immediate reactivity" while keeping their "antigenicity" [65]. After the discovery of T-cell's properties, allergists keep studying others chemical denaturation methods such as urea, as a way to reduce antigen's allergenicity while keeping its immunogenicity in order to originate safer products to "hyposensitization treatment" [66].

Chemical Polymerization:
The formalin's properties, earlier used to denaturize and precipitate pollen's extracts, lead to further studies that demonstrated that the formalized extracts have a high molecular weight due to chemical polymerization, originating the conceptualization of the allergoids [29]. In preliminary studies, the most promising agents to produce allergoids by chemical polymerization were the aldehydes, mainly formaldehyde and glutaraldehyde [67]. The aldehydes react with the primary amines, producing covalent cross-links between the proteins while preserving some of the immunological and enzymatic properties of the original extract [68,69]. This concept was largely explored since it was observed that polymerized allergoids were signi icantly less reactive than the native extracts, while retaining their capacity to induce blocking antibodies in guinea pigs [70]. Glutaraldehyde polymerized extracts of grass pollen and house dust mite signi icantly increased the ratio of regulatoryto-effector T cells in peripheral blood mononuclear cells (PBMC) cultures of allergic humans to these antigens when compared to the native extracts [71]. Meanwhile, in allergic volunteers, skin tests demonstrated the reduction on allergenicity produced by polymerization [72]. Clinical studies demonstrated the opportunity of chemically polymerized allergoids to be used as a tool for allergic desensitization [73]. The irst allergens submitted to chemical polymerization were the pollen's extracts, followed by mite's extracts and food allergens such as albumin [74][75][76][77][78][79][80]. Recently, glutaraldehyde polymerization is being accomplished also with cat dander [81]. Comparing the polymerization produced by the aldehydes, glutaraldehyde apparently had a stronger effect than formaldehyde, since its polymers are less allergenic, but possess also a lower T-cell stimulatory capacity [82]. The process of polymerization (chemical or enzymatic) may be facilitated by the use of additives, usually amino acids [29,83]. The polymerization process generates relatively great molecules, from the order of 10 5 to 10 6 Da [84]. This increase in the molecular weight prevents its absorption by the mucosal membranes, further systemic circulation and occasional allergic reactions [85]. This peculiarity turns the polymerized allergoids particularly attractive for use in sublingual preparations once the irst pass to tolerization is the internalization by phagocytosis/pinocytosis of the antigens by the submucosal DCs [86]. To increase the DC intake and subsequently improve the T-cell stimulatory capacity, glutaraldehydegenerated allergoids may be covalently coupled with mannan, a fungi polysaccharide with a pathogen-associated molecular pattern (PAMP) activity able to link to several pattern recognition receptors (PRRs) [87]. Now a day, chemically polymerized allergoids are commercially available to contribute with the tolerance-induction strategy in the treatment of allergic patients [88][89][90][91].

Carbohydrate conjugation
There are several simple techniques to couple carbohydrates to allergenic proteins and allergoids. The resulting neoglycoproteins loose several functional activities from the original molecules, such as enzymatic activity and IgE binding capacity. The coupling with preserved innate antigens such as mannan has the additional property of increase the capture of the molecule by the dendritic cells [92].

Enzymatic polymerization
Food industry has employed, for a long-time, cross-linking microbial enzymes as an addictive to increase the consistence of dairy products by means of polymerization [93]. The transglutaminase (TG) produced by the Streptoverticillium mobaraense is commercially available to produce culinary products either at a industrial level in the inalization of whey cream cheeses and yogurts, or at a handcraft level in Japanese restaurants where the chefs use it as an edible glue for foods [94]. One of the main activities of TG is the catalysis of the covalent link between the ε-amino group of lysine with the γ-carboxamide group of glutamine resulting in resistant intra-molecular and inter-molecular proteic bridges and polymerization [95,96]. Transglutaminase is a multifunctional enzyme present in most living organisms, including the human being [97]. Submitted to the bioavailability and immunoreactive tests recommended by the FAO/WHO to approve food addictives, the microbial TG was susceptible to peptic digestion and did not present amino acid sequences coincident with any known food allergen [98]. The modi ications induced by TG in the allergenicity and immunogenicity of bovine beta-lactoglobulin are yet at pre-clinical studies but have already demonstrated by in vitro, in vivo and ex vivo analyses promising results for the employ of TG polymerized food proteins as allergoids to treat allergic patients [40,[99][100][101]. The TG repolymerization of hydrolyzed fragments of cow's milk proteins also produced a great reduction on immunoreactivity [102].

Chemically modifi ed monomeric allergoids
Monomeric allergoids are allergens with punctual modi ications at strategic sites that do not produce polymerization and preserves the T-cell linear epitopes [103]. Mite, pollens, cat epithelia and food allergens have already been chemically modi ied in order to lower their capacity to bind speci ic IgE [104]. The most clinically employed method uses potassium cyanate (KCNO) to transform the lysine's ε-amino group into ureido-groups [105]. These carbamylated allergens demonstrated a lower in vitro and in vivo allergenicity while preserving immunogenicity as demonstrated by the capacity to induce IgG antibodies able to react with the native antigens [106]. Carbamylated allergoids have proved safe and effective in the treatment of allergic conditions [107]. This strategy is already commercially available, mainly in Europe as sublingual tablets for treatment of dust mite and/or grass-pollen allergic patients [108][109][110][111]. Other methods such as the succinylaton are yet at pre-clinical study [112].

Allergen peptide immunotherapy
The reduction of three-dimensional complex proteins to simpler linear peptides is the irst physiological gastrointestinal mechanism to reduce food protein allergenicity. However, alone, it is not always enough to provide a sustained tolerance to the dietary antigens [113]. The intracellular digestion that occurs in dendritic cells (DC) also promotes the destruction of the conformational epitopes and generates free linear epitopes presented to Treg cells to promote immune tolerance. Peptide immunotherapy can provide not only a hypoallergenic provision of T-cell linear epitopes to a toleranceinduction strategy but also a supply of short monovalent B-cell epitopes unable to produce cross-links between antibodies [114]. These peptides, able to bind to speci ic antibody's paratopes (competing with the original antigen's epitopes) are called "mimotopes" [115]. The saturation of the reaginic paratopes with monovalent mimotopes prevents the cross-link between reaginic antibodies ixed to the surface of the effector cells, blocks the signaling to the allergic reaction and produces a state of temporary competitive desensitization. That is also the mechanism explored during mast cell desensitization to treat drug hypersensitivity [116]. Aspirin desensitization is an example of mimotope desensitization. As a small molecule, with an average mass of 180Da, aspirin by itself is not able to cross-link antibodies, but when conjugated to circulating proteins it becomes an allergen. Aspirin desensitization is done by slow saturation of antibodies ixed at effectors cells surface, allowing a temporary state of tolerance [117]. Eventually the tolerogenic administration of a hapten may also induce a permanent state of tolerance if the resulting hapten-conjugate protein is able to induce cellular and/humoral mechanisms to block the hypersensitivity, as it is achieved in nickel desensitization [118,119]. Therefore, peptide immunotherapy may provide two distinct mechanisms to treat allergic patients. The irst mechanism is a tolerogenic Treg-orchestrated desensitization (tolerogenic desensitization) that generates tolerogenic cytokines (IL-10, TGF-β) as well B cell-induced blocking antibodies (IgA and IgG4). The second mechanism is a competitive mimotope desensitization (competitive desensitization) that competitively blocks the encounter of the original allergens with the reaginic antibodies preventing cross-linking and the subsequent allergic reaction [120]. Once proved that the allergenicity of the mimotopes are clinically insigni icant in a given patient, the concentration of the initial peptide immunotherapy may be relatively greater than the concentration of natural allergens as would be employed in an ASIT strategy. So, in a combinative AIT with allergoids and natural antigens, the participation of allergoids can be initiated with higher concentrations, followed by a gradual increase of the unmodi ied allergen concentrations. This strategy will explore the competitive desensitization promoted by the mimotopes along with an increasing stimulation of the Treg tolerogenic mechanisms. Besides allergen immunotherapy, mimotopes are tools also used for a great variety of applications such as epitope mapping and cancer immunotherapy [121]. Peptide immunotherapy may be accomplished in two ways: enzymatic hydrolysis and biological engineering.

Enzymatic hydrolysis
Peptic digestion is the irst physiologic barrier to prevent food allergic diseases [122]. Pepsin is a gastric protease that naturally renders tolerable most allergenic proteins that otherwise would produce allergic symptoms [123]. So do trypsin and others proteases and peptidases found in small intestine [124]. The in vitro use of a simulated gastric juice is an established method to study and predict protein's allergenicity, since the more resistant to peptic digestion, the greater the possibility to produce sensitization and allergy [125]. The oral allergy syndromes, induced by pepsin-sensitive allergens such as pollens and fruits demonstrate this concept, since they usually produce local symptoms before the gastric passage, but may occasionally produce systemic symptoms when the digestive function is compromised by some disease or by the use of antacids [126][127][128]. The hydrolysis produced by mean of the use of different proteases is a strategy to reduce allergenicity of clinically relevant proteins. A given protein may originate different hydrolysates according with the protease employed [129]. Additionally, the oral use of partially hydrolyzed (but not extensively hydrolyzed) proteins was able to induce tolerance to egg's and cow's milk proteins in animal models [130][131][132][133]. The tolerogenic effect is dependent of the presence of the T-cell epitopes inside the peptide sequence of the hydrolysate and related to the reduction of both serum histamine and IgE, the increased intestinal expression of TGF-β and FOXp3, as well the inhibition of both Th1-biased (IFN-γ and IL-12) and Th2-biased (IL-4 and IL-13) cytokines [134]. However, differently of the native proteins, a casein extensive hydrolysate alone was not able to stimulate a humoral or cellular response in human babies [135]. The association of allergoids produced by peptic digestion with unmodi ied allergens can also be used as a strategy to induce tolerance to pepsin-sensitive allergens [8].

Biological engineering
When designing an antibody-guided immunotherapy, the departure point is the choice of the best protective epitope. Diversely from the immunotherapies extracted from the natural sources presenting a great variety of immunogenic epitopes, the engineered immunotherapy will usually deal with just one or a few molecules to produce an epitope-based vaccine [136]. So, the choice of the immunotherapeutic peptides is crucial, mainly due to the high costs of the methodologies. According to the technique employed, Bioengineering produces two kinds of peptides: the synthetic and the recombinant. Several software tools based on matrix algorithms assembled from X-ray crystallography data were developed to aid the design of immunotherapeutic peptides [137]. This "in silico" approach helps to improve the prediction of class I and class II MHC epitopes before the benchwork [138]. Synthetic peptides have been investigated as vaccine candidates for over 40 years [139]. Bacteriophage-based techniques, such as phage display peptide libraries are also valuable tools for epitope mapping, identi ication, validation and production of mimotopes in suitable amounts for allergen immunotherapy [121].

Synthetic peptides
The design of a T-cell-speci ic reactive peptide begins with the mapping of T-cells from allergic patients. The irst technique for the sequential production of a designed synthetic peptide was developed by the Nobel Chemistry laureate Robert Bruce Merri ield in 1963 [140]. The irst method to assemble a synthetic peptide with the resolution of a single amino acid in order to be used as an epitope was developed in 1984 and gave birth to the epitope mapping and assemblage techniques in solid phase such as the Pepscan, as well puri ication techniques to assure a proper immunogenic molecule [141][142][143]. Regarding to their use in the immunotherapy strategies, allergen-derived peptides are classi ied in long, short and overlapping peptides. The greater the peptide higher is its immunogenicity and allergenicity. Unfortunately, small peptides may not elicit strong immune responses, what brings the need of immunogenic carriers and adjuvants for some of them [139]. Long synthetic peptides (over a hundred residues) were designed to reproduce the entire sequence of the allergenic protein and cover all possible T-cell epitopes. The use in clinical trials of long synthetic peptides in the treatment of allergic patients demonstrated to be safe (despite some mild immediate and late reactions), produced Th1-type immune deviation, allergen-speci ic IL-10 production and T-cell hyporesponsiveness [144]. The residual side effects of the long synthetic peptides stimulated the employ of short synthetic peptides. Successful proof-of-concepts were described in the treatment of cat-allergic and bee venom-allergic patients with short synthetic peptides that were able to down regulate the allergic response to the whole protein [145][146][147]. However, the administration of Fel d 1 short synthetic peptides (even with only 27 residues of amino acids) was able to produce immediate and late allergic symptoms in a small number of patients. It was demonstrated by ex vivo experiments, that under high dosage stimulation, allergen-derived short peptides act directly (in the co-stimulatory presence, but not by intermediation of APC) on T cells with multiple TCR. This direct action on TCR decreases the production of TH2 cytokines such as IL-4 but may be responsible for the Th1-biased late reactions [148]. This lead to the conception of the overlapping T-cell epitopes peptides: shorter peptides (16-17 residues) sectioned from the main epitope with overlapping sections encompassing the entire epitopes sequence. The administration of a mixture of 12 Fel d 1 overlapping peptides did not produce immediate allergic reactions, but was able to produce late asthmatic reactions in a dose-dependent fashion and further tolerance to subsequent injections. These late reactions were associated with cutaneous late-phase reactions to whole cat dander [149]. Contiguous overlapping peptides are also being investigated for birch pollen AIT [150][151][152].

Recombinant peptides -isoallergens
Genetic engineering, by means of diverse laboratory techniques is able to produce homologous and heterologous pieces of recombinant DNA (rDNA) with punctual alterations that produce proteins and/or peptides with modi ied properties from the natural source [153]. The detailed characterization of the DNA/RNA segment responsible for the transcription/translation of a speci ic allergen allows to the investigators to produce, with help of the Escherichia coli transcription apparatus, grams amounts of original and modi ied peptides in order to select hypoallergenic derivatives (isoallergens) to employ for modulation of allergic reactions [154]. The use of these modi ied peptides in asthma mice models produced a decrease in the in lammatory in iltrate that suggests a competitive desensitization, independent of an additional Treg tolerance induction with blocking IgG production provided by the long-term use [155]. Recombinant allergens may present several occurring isoforms. The IgE-binding capacity of these isoforms may differ, what result that some isoforms may be more allergenic, while some isoforms may be more immunogenic. One of the most studied isoallergens is the recombinant Bet v 1, the major allergen of birch pollen. The allergenicity and immunogenicity of natural and recombinant isoforms of Bet v 1 was evaluated by immunoblot, skin tests and T cell activation. The recombinant d isoform of Bet v 1 showed to be less allergenic and more immunogenic than the natural and the recombinant a isoform of Bet v 1 [156]. The higher immunogenic capacity of the d isoform of recombinant Bet v 1 was attributed to its natural trend to aggregation [157].

Carrier bound peptides
The use of small peptides for AIT is convenient to induce mimotope competitive desensitization, but their immune hyporesponsiveness may not induce proper Treg orchestrated tolerogenic desensitization. To solve this problem some investigators coupled epitope-based small peptides with greater molecules that acted as a carrier to stimulate the innate processing of the peptide and a proper presentation to naive T cells in a tolerogenic environment. One of these carriers was the keyhole limpet hemocyanin, a metalloprotein that bounded to Der p 2 peptides induced speci ic IgG antibodies that inhibited allergen-induced basophil activation in a similar way that the native allergen, without the inconvenience of the side-effect produced by IgEmediated allergic reactions [158]. In a similar way the B subunit of the cholera toxin conjugated with ovalbumin, administered prior to the sensitization, lead to a decrease of antigen-speci ic IgE and an increase of antigen-speci ic IgG2a when compared to non-pretreated sensitized animals [159]. Another interesting fusion was the couple of grass pollen's epitope-based peptides with the PreS proteins from Hepatitis B virus (HBV). This conjugate was produced in large amounts by recombinant expression in Escherichia coli and when applied to animals and humans was able to induce not only allergen-speci ic IgG antibodies, that inhibited the patient's IgE binding to the grass pollen allergens, but also HBV-neutralizing antibodies [160].

Hybridization
Also, through genetic engineering, by means of Polymerase Chain Reaction (PCR) researchers are able to combine heterologous T cell epitopes from similar allergens into a unique hybrid molecule suitable for use in AIT. The engineered hybrid allergoid may be projected to have a peculiar conformational structure facilitating the uptake and processing by the dendritic cell, along with an insigni icant allergenicity allowing its use in tolerance-induction strategies to treat polysensitized patients [161].

Adjuvants
Adjuvants are substances aggregated to an AIT strategy that modify the way the antigens are processed by the immune system [162]. Adjuvants may act as immune stimulants increasing the innate processing of the antigen (dendritic cells target adjuvants), or may have just a depot-effect (adsorptive adjuvants), simply increasing the permanence of the immunotherapeutic agent on the site of the injection allowing more time for the innate immune system to process the antigens. Sometimes these effects are associated and inseparable. The primary function of an adsorptive adjuvant is not just in luence immunocompetent cells, but it is rather intended to exert a short-term or long-term depot effect, reducing the bioavailability of the subcutaneous allergenic extract. This effect is produced by precipitation of the antigen extract that slows its release and allows the use of fewer shots, optimizing the immunotherapeutic effect of each injection. Some adjuvants are employed by their intrinsic immunogenicity, such as the toll-like receptors (TLR) agonists that stimulate the tolerogenic dendritic cells and increase the internalization of the immunotherapeutic extract and further interaction with T-reg cells [163][164][165]. Among them, the C-type lectins ligands are the most attractive due to their role in allergic sensitization [166]. Lectins are a ubiquitous class of protein domains that share the property to bind to carbohydrates. The denomination C-type lectin refers to the lectins that need calcium to bind to the carbohydrate. Lectins may be found free on the interstitial milieu, where they act as opsonins, or anchored into cytoplasmic membranes where they work as innate receptors [167]. Dendritic cells may be therapeutically targeted with C-type lectins ligands to improve the internalization of the antigens of the AIT preparation [167]. Different adjuvants associated with the same antigen may induce different innate and adaptive immune responses, as well different reactogenicity patterns [168]. Adjuvants may be associated in formulations, especially between adsorptive and dendritic cells target adjuvants, what is now known as Adjuvant Systems [169].
Freund's Adjuvant: Freund's adjuvant was originally conceived as a water-inoil emulsion where the inactivated microorganisms were dispersed to stimulate the innate processing of their antigens. It may be optionally potentialized with mannide monooleate, an emulsifying agent, or with heat-killed mycobacteria, when it is called the Freund's complete adjuvant [170]. It is not used in human vaccines due to local in lammatory side effects.
Bacterial Lysates: Bacterial lysates inactivated by mechanical, thermal and/or chemical treatment lose their toxicity, but preserve their immunogenicity, acting as immunostimulants. They induce synergistic TLR innate immunity, a shift to production of Th1 cytokines such as type I interferons and Treg and Breg anti-in lammatory cytokines, such as IL-10, that attenuate Th2 allergic responses [171][172][173][174].
Aluminum: Salts of aluminum have been employed as immune stimulants since 1924, when it was demonstrated that they potentialized the production of horse's antibodies against the tetanus and diphtheria toxoids [175]. Their use in the treatment of allergic patients was irst described in 1938 [176]. The association of aluminum salts as an AIT adjuvant increases the uptake of the allergens by the antigen-presenting cells and increases the antigen-induced T cell proliferation [177]. To prepare aluminum salts precipitates (alum) for human use it is required a rigorous quality control because minor alterations on particle sizes can modify their properties. Alum was extensively used as a long-term depot-type adjuvant for several decades, however, despite some well-tolerated alum-depot allergen immunotherapies are yet in use; its employ is being gradually abandoned due to reports of toxicity as well immediate and delayed sensitivity [178][179][180][181][182][183][184].
L-Tyrosine: L-tyrosine is a non-essential amino acid synthesized in the human body from phenylalanine. L-tyrosine binds non-covalently to the allergenic extract burying its hydrophilic side into the protein's core, resulting in a hydrophobic derivative [185]. L-tyrosine acts in AIT as a short-term depot-forming adjuvant, decreasing the bioavailability of the formulation, resulting in an indirect increase of the IgG antibody production [186]. The addition of microcrystalline tyrosine to allergoids associated with TLR agonists provides a depot-effect to the AIT formulation [187]. Several different brands of allergoids adsorbed onto L-tyrosine are already in use in Europe for at least 3 decades [188,189].
Mannan: Mannan is a ubiquitous mannose polysaccharide found in plants and fungi that acts as a C-type lectin ligand. The innate immune system has patternrecognition receptors for mannose-terminated sugars that once activated enhance antigen presentation, DC maturation and immune responses [190]. The addiction of nonoxidized mannan to the glutaraldehyde polymerization of pollen allergens originated a stable allergen-mannan conjugate with low IgE-binding capacity further demonstrated to be a high immunogenic molecule, increasing the production of tolerogenic cytokines, being captured more ef iciently by human DCs than the nonconjugated allergoids and the native allergens [191].

Monophosphoril Lipid A: Monophosphoryl lipid A (MPLA) is a hydrolyzed lipid
A fraction derivative from lipopolysaccharides (LPS) of the outer membrane of Gramnegative bacteria that works as a TLR-4 agonist [192,193]. The main characteristic of MPLA is its strong immunostimulatory properties and absence of endotoxic effects [194]. This safety/ef icacy pro ile allowed the use of MPLA as an adjuvant incorporated into several commercial vaccines, including AITs with polymerized allergoids [195][196][197][198][199][200].
Virus-like particles: The innate immune system evolved to recognize viral particles by PRRs such as the TLR-7, that when activated by single stranded viral RNA stimulates the production of Th1 cytokines such as the type 1 interferons, inhibiting the Th2 pro ile. This property may be advantageously explored in the treatment of type-1 allergies employing virus-like particles in association, or even in the absence of speci ic allergens (non-speci ic desensitization). Preclinical trials with a TLR-7 agonist administered by nasal route demonstrated a reduction in the nasal score symptoms after nasal allergen challenge. The main collateral effects were systemic in luenza-like symptoms like fever and myalgia [201].
DNA immunostimulatory sequences (CpG): Unmethylated Cytosine-phosphodiester-Guanine (CpG) motifs are TLR-9 ligands that conjugated with bacterial and virus antigens activated Th1 cells, cytotoxic T cells (CTL) and induced the production of gamma-interferon (IFN-γ) and IgG neutralizing antibodies [202]. The conjugation of the ragweed-pollen antigen with DNA immunostimulatory sequences rich in unmethylated CpG motifs, resulted in an immunotherapeutic compound with low allergenicity and increased immunogenicity [203]. These oligodeoxyribonucleotides conjugated with Amb a 1 increased the allergic human PBMC production of Th1 cytokines and decreased the Th2 expression, demonstrating a great immunotherapeutic potential for allergic diseases [204]. Pilot studies employing as AIT strategy injections of conjugates of house dust mite (HDM) or Amb a 1 with immunostimulatory oligodeoxyribonucleotides in allergic patients inhibited the cutaneous and conjunctival responses to sensitizing extracts and alleviated allergic symptoms with good tolerance [205,206].
Lysine Analogs: Lysine analogues act as competitive inhibitors of protein interactions that depend on the lysine residues [207]. Actually, they are indicated for treatment of bleeding disorders due to its competitive suppressing of plasminogen activation by blood and tissue kinases [208]. The ε-aminocaproic acid (an analogue of lysine without the α-amino group) was already used empirically as an adjuvant of SLIT [209]. Theoretically, it has the potential to impair the liaison of the IgE with the lysinedependent binding sites of the immunotherapeutic extract, lowering its allergenicity. It was extensively used in Brazil for more than 30 years, since the 70's with the brand name of Aminovac®, commercialized by drugstores under medical prescription. In the early 2010's it was withdrawn off the market by the sanitary authorities with the explanation that the producer did not ful ill the novel requisites to renew its license.

Vitamin D3
When comparing the capacity of polymerized versus native allergen extracts of grass pollen and HDM to stimulate the proliferation and cytokine production in PBMC of allergic volunteers, the addiction of 1α,25-dihydroxyvitamin D3 reduced effector T cell activation and cytokine production [71]. Further studies demonstrated that Vitamin D3 potentiated the effect of a chemically-modi ied monomeric allergoid in sensitized mice [210].

Epigenetic Inducers of Foxp3
The expression of FoxP3 in Treg cells depends on DNA demethylation at a conserved CpG-rich region in the FoxP3 locus [211]. DNA methylation is mediated by DNA methyltransferases (DNMTs) which is inhibited by the micro-RNAs encountered in the fat of human breast milk and unprocessed cow's milk [212]. The milk-derived immune-regulatory exosomal micro-RNAs, particularly rich in the irst months of lactation produce demethylation of the FoxP3 locus activating Treg cells [213]. Similarly the intake of raw cow's milk is also associated with high FoxP3 demethylation and differentiation of Treg cells [214]. The use of FoxP3 inducers is yet just an speculative matter in the ield of AIT adjuvants, but justify the incentive to the use of breast milk as prophylaxis for allergic diseases [215].

Conclusion
The science behind the creation of strategies to induce protective immune responses in patients with allergen-induced hypersensitivity reactions had evolved a lot. More than a century of studies revealed many promising approaches that had bene iced an uncountable number of allergic patients. However, most studies and strategies are directed through Gell & Coombs type I hypersensitivity mechanisms which diagnosis is easier and simpler than the other three hypersensitivity reactions. However, the understanding that cellular hypersensitivity precedes the Th2 escalation and that the AIT therapeutic effect does not act directly over IgE, but rather over cellular mechanisms of tolerance, lead us to the believe that the other hypersensitivity mechanisms may also be modulated through AIT [216,217]. Therefore, there is yet a long way to a better understanding of the tolerance mechanisms and the discovery of the ultimate therapy. Meanwhile, the allergic population grows worldwide, requesting a personalized, simple, affordable and quick cure for these debilitating conditions.