Past, present, and future of allergen immunotherapy vaccines

Abstract Allergen‐specific immunotherapy (AIT) is an allergen‐specific form of treatment for patients suffering from immunoglobulin E (IgE)‐associated allergy; the most common and important immunologically mediated hypersensitivity disease. AIT is based on the administration of the disease‐causing allergen with the goal to induce a protective immunity consisting of allergen‐specific blocking IgG antibodies and alterations of the cellular immune response so that the patient can tolerate allergen contact. Major advantages of AIT over all other existing treatments for allergy are that AIT induces a long‐lasting protection and prevents the progression of disease to severe manifestations. AIT is cost effective because it uses the patient´s own immune system for protection and potentially can be used as a preventive treatment. However, broad application of AIT is limited by mainly technical issues such as the quality of allergen preparations and the risk of inducing side effects which results in extremely cumbersome treatment schedules reducing patient´s compliance. In this article we review progress in AIT made from its beginning and provide an overview of the state of the art, the needs for further development, and possible technical solutions available through molecular allergology. Finally, we consider visions for AIT development towards prophylactic application.


| INTRODUC TI ON
IgE-associated allergy, the most common immunologically mediated hypersensitivity disease, is based on the formation of IgE antibodies against per se harmless and mainly environmental antigens, termed allergens. 1 Subjects with a genetic predisposition for allergy (ie, atopic subjects) produce IgE antibodies against allergens in their environment. 2 IgE binds to mast cells and basophils via high-affinity receptors for IgE so that subsequent allergen contact can induce mast cell and basophil activation by cross-linking of cell-bound IgE. This leads to release of inflammatory mediators and cytokines and thus immediate allergic inflammation. 3,4 Antigen-presenting cells, especially B cells and dendritic cells can bind IgE via the low-or high-affinity receptor for IgE, and via IgE-facilitated allergen presentation cause T-cell activation and secretion of inflammatory Th2 cytokines leading to activation of eosinophils and formation of innate Th2-like immune cells such as group 2 innate lymphoid cells (ILC2s). 5,6 In contrast to allergic patients, nonallergic subjects produce allergen-specific IgG antibodies without experiencing allergic inflammation upon allergen contact. 7,8 While anti-inflammatory treatment based on pharmacotherapy and biologics which neutralize allergen-specific IgE or inflammatory cytokines can ameliorate allergic inflammation, only AIT represents a causative treatment. 9 In fact, AIT induces a protective immunity in allergic patients consisting of allergen-specific IgG antibodies which serve as "blocking antibodies". They prevent IgE from binding to the allergens and thus the complete consecutive downstream cascade of allergic inflammation induced by IgE allergen immune complexes. 10,11 AIT also profoundly affects allergen-specific cellular responses which may be also due to the ef-  12,13 A study by grass pollen immunotherapy has shown a significant decrease in memory TFH cell numbers after immunotherapy. 14 Additionally, TFR cells were found to produce more IL-10 compared with TFH cells. A possible plasticity between TFH and TFR cells has been suggested in the same study, indicating that TFR cells may play important roles in suppressing TH2 responses during AIT. 14 IL-10-secreting allergen-specific Breg cells which may be capable of suppressing allergen-specific CD4 + T cells and producing allergen-specific IgG4 antibodies have been identified in bee venom-tolerant beekeepers and patients having received venom AIT 15 as well as in house dust mite allergen immunotherapy. 16 Additionally, Breg cells may have inhibitory capacity by producing IL-35 and TGF-β. 17 Apart from Treg and Breg cells, IL-10-secreting natural killer regulatory cells have also been shown to suppress allergen-stimulated T-cell proliferation in humans and may be important in tolerance induction as other regulatory cell types. 18 All these aspects and their relevance for AIT are currently being investigated.
Major advantages of AIT are that, conceptually, AIT is a therapeutic vaccination which induces a protective allergen-specific immune response. Only small amounts of the disease-causing allergen or allergen derivatives are needed for generating and maintaining the protective immune response. Like other vaccination approaches, costs for treatment are affordable. 19 Usually, after 3 years of AIT treatment beneficial effects continue for a few years, even when AIT has been discontinued. 20 These long-term effects may be attributed to the persistence of high-affinity and functional allergen-specific IgG 4 antibodies. [21][22][23] However, boosting of antibody responses may become necessary after discontinuation. One of the most important aspects of AIT is that it seems to halt the progression of mild symptoms toward severe symptoms as has been demonstrated in children. 24 In fact AIT, but not pharmacological treatment prevented the progression of allergic rhinitis toward asthma.
However, there are also some major problems which prevent the broad application of AIT. 25 First of all, AIT is a form of precision medicine which requires that the disease-causing allergens are identified and the correct vaccine is administered. Second, polysensitization against many different allergens requires that for each of these allergens effective and safe vaccines are available and can be co-administered. Third, administration of allergen to patients by AIT can cause side effects. Therefore, very cumbersome treatment schedules starting with tiny doses up to maintenance doses and multiple administrations make the treatment inconvenient for patients leading to low patients' compliance. Finally, the quality of natural allergen extracts represents a major bottle neck for producing safe and effective AIT vaccines. 26 Additional issues are to define the optimal time to start AIT, dosing as well as intervals, when to stop/continue AIT, and there is a need for biomarkers. Therefore, we will review briefly some important milestones in the evolution of AIT before we discuss current AIT and its unmet needs.

| THE IMP ORTAN CE OF THE PA S T OF AIT
The famous quotation of Confucius, the Chinese philosopher and reformer (551 BC-479 BC), "Study the past if you would define the future" indeed applies very much for AIT because one will then realize that important discoveries have been made long ago and that a K E Y W O R D S allergen, allergen-specific immunotherapy, allergy, molecular allergy vaccines continuous improvement along these milestones will allow us not only to improve AIT but eventually also to use the AIT treatment concept for specific prevention of allergy. Therefore, we will have a look into the historic development of AIT.

| Some milestones in the development of AIT
In 1911, when Leonard Noon published the first study showing that injection of pollen extract improved symptoms of grass pollen allergy, the immunological basis of hypersensitivity to grass pollen was not known. 27 At that time, neither IgE antibodies nor allergens had been characterized and the assumption was that pollen contained a toxic substance which is responsible for the inflammatory reaction.
The obvious question was why Noon considered active immunization with a "toxic" component which per se would induce a toxic reaction. The answer can be found in Noon's original paper where he refers to earlier work published by Dunbar in 1903 who had shown that antisera raised in animals against pollen toxin could neutralize this "pollen toxin". 28 Thus, Noon considered that it may be possible to induce an "anti-pollen toxin" immune response also by active immunization. In Table 1 we have listed some of the important milestones in AIT in a chronologic order and provide for each of those the corresponding references.
It thus becomes clear that the first AIT studies including the ragweed SCIT study by Clowes 29 were performed before fundamental mechanisms of immediate-type hypersensitivity were established. it demonstrated that immediate-type hypersensitivity can be transferred by a serum factor which then was defined as reagin (ie, IgE antibodies) and was specific for a certain antigen (ie, allergen). In addition, a tissue component which is present in allergic as well as nonallergic subjects (ie, mast cells) was needed for an immediate-type allergic reaction. The study by Prausnitz & Küstner was also important because it provided researchers with an experimental system (ie, Prausnitz-Küstner reaction) which could be used to search for allergen-specific sensitization by in vivo testing and opened the possibility to identify IgE antibodies as the key serum factors in allergic reactions. 31,32 Key topics in AIT were the following: First, there were several mechanistic studies which indicated that the induction of allergen-specific IgG blocking antibodies is an important mechanism in AIT. The study by Dunbar conducted in 1903 demonstrated that allergic reactions can be specifically prevented with anti-sera raised against allergen preparations already before the first AIT study was conducted by Noon. 28 In 1935 Cooke published a seminal paper showing that allergen-specific skin reactivity can be suppressed by post-AIT sera using the Prausnitz-Küstner reaction in human subjects. 33 Loveless then identified allergen-specific IgG antibodies as the serum factor responsible for the suppression of allergic reactions. 34 The original experiment conducted by Dunbar was confirmed by the demonstration that allergy can be also treated with human hyper gamma immunoglobulin. 35 Finally, a recent study demonstrated that immediate symptoms of cat allergy can be prevented by the administration of recombinant human IgG 4 antibodies specific for the major cat allergen, Fel d 1, confirming that blocking IgG antibodies are of key importance for suppression of allergic symptoms. 36,37 Second, major attempts were made to improve the quality and specificity of AIT as well as the safety of AIT by decreasing side effects. In this context the study by Sledge needs to be mentioned which demonstrated that the adsorption of allergens onto the adjuvant Aluminum hydroxide prevented the systemic release of allergens in the body and thus reduced severe systemic anaphylactic side effects. 38 It was then noted that allergen fragments obtained by isolating low molecular weight material from allergen extracts or by digestion exhibited reduced allergenic activity. 39 Also plasmid DNA vaccination was tested in experimental animal models and has been used in a first AIT trial recently. [48][49][50] The conjugation of allergens with immunomodulatory DNA sequences represented another strategy to reduce the allergenic activity of the AIT vaccine and to have immunomodulatory function. 51 Carrierprotein bound allergen-derived B-cell epitope peptides represent the newest generation of recombinant hypoallergenic allergen derivatives which have entered clinical trials. 52,53 The grass pollen allergy vaccine BM32 was not only found to be nonallergenic but also induced strong allergen-specific IgG responses. It also did not boost allergen-specific IgE production and hence may be considered for prophylactic vaccination. 53 In addition to modifications in AIT vaccines to be more specific and safe, different application routes have been explored with a view of making AIT more convenient. Early oral immunotherapy (OIT) dates back at least to 1927 and currently is tested for various forms of food allergy. 54

| Can allergen extract-based AIT be improved?
All of the AIT vaccines which are available today are based on allergen extracts which are obtained from natural allergen sources. There are huge differences among allergy vaccines from different companies and also variations from one batch to another batch produced by a given manufacturer depending on the quality and purity of the raw materials, the methods of extraction and the representation of the individual allergen molecules and their immunogenicity. 26 Today most drugs and biologics (eg, blood coagulation factors, protein/ peptide hormones, cytokines) are produced by recombinant expression, partly because of low abundance in natural sources, and purification of natural components belongs to the past. However, AIT vaccines are still produced from natural allergen sources, although today almost all relevant allergen molecules can be easily produced by recombinant expression. 25,63 In the meantime, multiple studies document that the quality of natural allergen extracts (eg, grass pollen, birch pollen, house dust mites, dog, Alternaria) is poor. [64][65][66][67][68][69] For example, it has been shown that important allergen molecules are lacking in certain extracts, that the ratios of different allergens vary greatly, and that natural extracts can be contaminated with unwanted materials. 70 The molecular analysis of allergen-specific immune responses of patients undergoing AIT with natural allergen extracts has revealed that protective IgG antibodies are not induced against all important allergens. 71 It was shown that IgE sensitizations can be induced against allergens against which the patient was not sensitized before 72 and that incomplete induction of protective IgG responses is associated with less favorable clinical outcome. 73 The analysis of availabilities of AIT vaccines which have been evaluated in clinical studies sufficiently enough to provide evidence for efficacy and to follow the current rules for registered drugs has shown that only few AIT vaccines fulfill the current requirements for drugs. 26 The majority of AIT vaccines will therefore only be available on a named patient´s basis without the current documentation of efficacy and safety required for registered drugs. Attempts to obtain registration for allergen extract-based AIT vaccines by conducting the necessary large clinical trials up to phase III have been often not successful. One recent example is the failure of a subcutaneous birch pollen allergy vaccine in a phase III study 74 which did not reach its endpoints.
The limitations of allergen extract-based technologies are also evidenced by a "new" SCIT approach which is based on an extract from Lolium perenne pollen which was then hydrolyzed to reduce its allergenic activity. The hydrolyzed extract containing peptides of a size between 1 and 10 kDa was then used for SCIT without any adjuvant. In the first published studies the authors reported a modest induction of grass pollen allergen extract-specific IgG 75 and a reduction in allergen-induced conjunctival inflammation as determined by conjunctival provocation test (CPT). 76,77 Although a hydrolyzed grass pollen extract was used with the goal to reduce allergenic activity of the vaccine, an immediate-type grade IV reaction requiring epinephrine was observed in the large randomized, multicenter, double-blind, placebo-controlled trial. 77 In a selected patients population it appeared that the hydrolyzed Lolium perenne grass pollen vaccine is clinically effective 75 but unfortunately the clinical results of the phase III trial were disappointing. 78 Besides the recent approach of using hydrolyzed allergen extracts, several other attempts have been made to improve allergen extract-based AIT approaches. One major focus has been to test different routes of administration ( Figure 1). Besides subcutaneous AIT (SCIT) for which the underlying mechanisms are established best and which is used for the longest time, actually since 1911, also other routes of administration have been considered.

| Sublingual immunotherapy
Without doubts the greatest effort in terms of investment, development, and clinical trials has been dedicated to sublingual allergen administration in the last 30 years which has been summarized in a recent review article in a comprehensive way. 79 The first attempts of sublingual immunotherapy go back to the study of Scadding & Brostoff in 1986 55 and a first randomized controlled trial was published in 1998 by Passalacqua and colleagues. 56 Sublingual immunotherapy (SLIT) was thought to reduce side effects and thus to increase the ease of use of AIT by self-medication. Many controlled studies indicate that SLIT is clinically effective but until now the underlying mechanism is not fully understood because the treatment induces only very modest allergen-specific IgG responses and paradoxically increases allergen-specific IgE responses strongly. 80 Surprisingly, the ability of SLIT to reduce immediate allergic symptoms as measured by controlled in vivo provocation testing such as SPT has not been studied extensively. Experts have pointed out that it is difficult to calculate the placebo effect in AIT in general 81 and specifically in SLIT 82 even in placebo-controlled studies because it is difficult to blind for local reactions at the application site.
Nevertheless, SLIT approaches have been evaluated in multiple clinical studies and clinical efficacy has been demonstrated in several phase III studies. 79 However, real-life-compliance for SLIT is much lower than that for SCIT. 83 After 3 years of treatment less than 7% of SLIT patients adhere to treatment. Furthermore, for many important allergen sources, such as venoms, food allergy, and many respiratory allergen sources, such as cat dander and molds, no effective SLIT treatments are available.

| Oral immunotherapy
Another alternative approach to SCIT is oral immunotherapy (OIT).
After the early OIT reports 54 this approach has been tried for respiratory allergen sources but was found not to be effective. 84

| Intralymphatic immunotherapy
As another alternative for SCIT, it has been suggested to perform ILIT was found to be acceptable but a disadvantage of the treatment is that is requires ultrasound guidance to deliver the vaccine into the lymph nodes which makes it quite cumbersome. As compared with other routes relatively few studies have been performed so far mainly with allergen extracts and few with defined purified allergen derivatives. 57

| Epicutaneous immunotherapy
More than 10 years ago the first studies appeared, suggesting epicutaneous allergen administration for epicutaneous immunotherapy (EPIT). 91,92 An overview of experience gained in EPIT trials is summarized in a very recent review article. 93 This review concludes that "EPIT might induce desensitization in peanut allergy and an increased risk of local adverse events (AEs). These findings should be interpreted with caution owing to the limited study and heterogeneity.
More data in the older (children ≥ 12 years and adults) and other allergic diseases are needed".
In fact, a detailed study of systemic allergen-specific antibody, T-cell, and cytokine responses after epicutaneous allergen administration has indicated that it induces only very modest systemic IgG increases and a detectable activation of allergen-specific T-cell responses. 94 In fact, the analysis of systemic peanut allergen-specific IgG responses in the course of EPIT has confirmed these findings.
A very low induction of IgG responses only to certain peanut allergens was noted. 95 The idea behind EPIT is that allergen administra- EPIT, and SLIT each has its unique pros and cons". 99

| Short comparison of different routes for AIT
A short comparison of AIT routes as displayed in Figure 1

| Clinical experience with new adjuvants
It thus appears that the use of alternative routes cannot overcome the limitations set by the quality of natural allergen extracts. However, attempts were also made to improve SCIT by using alternative adjuvants. The type-B immunostimulatory phosphorothioate oligode-

| MOLECUL AR IMMUNOTHER APY FOR FUTURE TRE ATMENT AND ALLERG EN -S PECIFI C PRE VENTI ON
The era of molecular allergology started with the isolation of the DNA-encoding major allergens more than 30 years ago. [105][106][107][108] Already shortly after the expression of the first recombinant aller-

| Molecular allergy diagnosis for prescription and monitoring of AIT
The concept of using allergen molecules as diagnostic tests for prescription and monitoring of AIT was presented the first time at the Meeting of the European Academy for Allergy and Clinical Immunology (EAACI) held 2002 in Naples, Italy. 115 Shortly thereafter micro-arrayed allergens were developed and proposed for the same purpose. 112 The fundamental idea behind the use of allergen molecules was that allergen sources contain source-specific allergens and cross-reactive allergens. IgE reactivity to the sourcespecific allergens serves as an indicator that the patient is genuinely sensitized against a particular allergen source. On the other hand IgE reactivity with highly cross-reactive allergens was considered helpful to discriminate genuine sensitization from cross-sensitization.
Thus, the availability of molecular testing was suggested to assist in the selection of correct allergy vaccines. In the meantime this concept was found to be very helpful for the correct prescription of AIT as has been demonstrated in several clinical studies thereafter. [116][117][118][119][120][121] Thus, molecular allergy diagnosis is well established as a companion diagnosis to guide the prescription of AIT and for the monitoring of the effects of AIT. 122 It is currently used as companion diagnosis for allergen extract-based AIT but will be even more useful for molecular AIT approaches. Furthermore, molecular allergen reactivity profiles as determined for different countries and populations are important for the selection of allergen molecules to be incorporated into new molecular AIT vaccines. 7,123,124

| Molecular AIT approaches
The different molecular approaches for AIT are displayed in Figure 2.
In fact, everybody would have expected that the use of native-like recombinant allergens mimicking the immunological properties of the natural allergen molecules would have been the first approach for molecular AIT. However, the molecular era of AIT started with attempts to treat patients with short, non-IgE-reactive, allergenderived T-cell epitope-containing peptides followed by recombinant hypoallergenic allergen derivatives with reduced IgE reactivity before first AIT trials were conducted with native-like recombinant allergen molecules. The reason for this may have been that investigators were keen to improve immediately two aspects of AIT, specificity by using defined molecules and safety by using allergen derivatives which were designed to have reduced allergenic activity.

| T-cell epitope-containing peptides
Allergen-derived T-cell epitope-containing peptides are synthetic peptides comprising T-cell epitopes of allergen lacking IgE reactivity ( Figure 2). The goal of injecting such peptides into patients was to induce T-cell tolerance and it was hoped that this would reduce allergen-specific IgE production and allergen-induced inflammation. 126 The approach was started already soon after the first allergen DNAs were isolated using Fel d 1, the major cat allergen as a model. 44,45 However, the first trials were not successful and even when the approach was refined more recently, peptide-based AIT was not effective. In fact, in a phase III trial involving more than 1000 patients a huge placebo effect was observed and active treatment did not achieve improvement over placebo. 127 The T-cell epitope-containing peptides are relatively short and, accordingly, this treatment did not induce allergen-specific protective IgG antibodies which may be one reason for the failure of this approach. Although the T-cell epitopebased approach was not successful for the treatment of patients with allergy, it is quite possible that this strategy may be useful for the induction of T-cell tolerance in a preventive approach but this has not yet been studied in clinical trials.

| Recombinant and synthetic hypoallergens
After the T-cell epitope-containing peptides, recombinant hypoallergenic allergen derivatives were the first to be evaluated in clinical AIT studies in patients. 62 They do not elicit immediate side effects, but upon immunization, induce allergen-specific IgG antibodies to a various extent (Figure 2).
Allergen-specific T-cell epitopes remain largely preserved in these molecules and hence may give rise to late-phase, T-cell-mediated side effects. Available proof of principle AIT studies in patients demonstrate that hypoallergens are clinically effective. 25 Furthermore, preclinical studies demonstrate that hypoallergens induce blocking IgG antibodies and can be developed for many respiratory, venom, and also food allergens. [133][134][135][136][137] In fact, recent results of a I/ IIa phase double-blind, placebo-controlled clinical study (Identifier: The importance of the two studies with native-like recombinant allergens is that they demonstrate the equivalence and even advantages of recombinant allergens over natural allergen extracts.
Native-like recombinant allergens could therefore be used instead of natural allergen extracts and would offer the advantage to be well-defined and to contain all relevant epitopes. SCIT with these molecules induces allergen-specific blocking IgG. However, like natural allergens they induce immediate-type and late-phase side effects because of preserved IgE reactivity and T-cell epitopes.
Accordingly, SCIT with native-like recombinant allergens requires the same cumbersome up-dosing schedules and multiple maintenance injections as SCIT with natural allergen extracts. However, the advantage over natural allergen extracts is the high quality of the vaccine because allergens can be produced at low cost in a defined and reproducible manner. In this context, also a recent successful SCIT study using purified natural major Alternaria allergen Alt a 1 should be mentioned. 142 As recombinant Alt a 1 was found to be equivalent with natural Alt a 1 one may assume that this could have been equally achieved with purified rAlt a 1.

| Allergens coupled to immunomodulatory compounds
CpG oligodeoxynucleotides were found to be useful as adjuvants to induce Th1 immune responses and their immunomodulatory activity in a murine model of asthma was demonstrated. 143 Researchers from the company Dynavax which had pursued nucleic acid-based AIT approaches found that coupling of such immune-stimulatory DNA to the major ragweed allergen Amb a 1 enhanced its immunogenicity and at the same time reduced its allergenicity. 144 Based on this finding a SCIT trial was conducted with this vaccine showing that it induced allergen-specific blocking antibody responses and led to a reduction in allergen-specific IgE levels. 51 The vaccine also showed beneficial clinical effects but the approach was not further pursued because the outcome of subsequent studies was less favorable and the production of the vaccine by chemical coupling turned out to be challenging.

| Virus-like particle-coupled allergens
The first generation of virus-like particle-coupled allergens followed a similar principle as described for allergens coupled to immunomodulatory sequences. Allergen molecules were coupled chemically [145][146][147] and also by specific linker systems to virus-like particles produced by recombinant expression. 148 As a result, a reduced allergenic activity of the vaccines and good immunogenicity were noted. A major hurdle for the virus-like particle coupled allergens for further clinical use is that it is very difficult to produce these vaccines in a reproducible manner due to the coupling process which is difficult to control. It is quite possible that the reduced allergenic activity of these vaccines is achieved not only by "hiding" of IgE epitopes by the VLPs but also by the formation of large oligomers as was observed for a recombinant Bet v 1 trimer 149

| Allergen-encoding nucleic acids
More than 20 years ago, two experimental studies were published, indicating that it is possible to vaccinate with allergen-encoding DNA to obtain an allergen-specific Th1 response and allergen-specific IgG responses in murine models. 48,49 This finding was important because it indicated that DNA vaccination for AIT may be possible. 157 Major challenges of this approach were to prevent uncontrolled release of allergen in the vaccinated person and thus the risk of allergic side effects. To this end DNA vaccination with DNA coding for hypoallergens and RNA vaccination which has a reduced risk of generating relevant allergen amounts in the tissues were considered. 158 A first clinical study reporting DNA vaccination has been published, 50 but no information is available if this strategy induces a protective immune response.

| Carrier-bound B-cell epitopecontaining peptides
Carrier-bound B-cell epitope-containing peptides represent a further development of recombinant hypoallergens. 138,159 The currently most advanced platform uses hepatitis B-derived PreS protein as a carrier protein to which nonallergenic peptides derived from the IgE-binding sites of allergens are fused. The vaccines are made as recombinant fusion proteins by expression in Escherichia coli by a well-controlled procedure delivering large amounts of the fusion proteins in consistent quality. [160][161][162][163] The hypoallergens are characterized by lack of IgE reactivity and hence do not induce any immediate side effects but blocking IgG antibodies upon immunization. 164 Allergen-specific T-cell epitopes are largely replaced by the carrier molecule which reduces late-phase, T-cell-mediated side effects.
Importantly, the grass pollen allergy vaccine BM32 which has been constructed according to this principle has been tested in several clinical trials and was found to be safe and to induce beneficial clinical effects even in double-blind, placebo-controlled multicenter field studies. 52,53,137,165 BM32 was found to induce continuously grow- technology may be also applicable for the production of AIT vaccines against venom and food allergens. BM32 has undergone three phase II studies and a phase III study is currently planned to make the vaccine ready for registration.

| Recombinant allergen-specific IgG blocking antibodies
The demonstration of Dunbar that allergen-specific antisera can prevent allergic reactions in vivo 28 and the study by Cooke showing that AIT-induced IgG can suppress allergen-induced immediate-type skin reactions 33 indicated that the induction of allergen-specific IgG antibodies is a major mechanism of AIT. However, in order to obtain defined vaccines for passive immunization, human allergen-specific blocking antibodies are needed. More than 20 years ago the first publications appeared in which such allergen-specific antibodies were reported. One study reported human monoclonal IgA antibodies specific for the major ragweed allergen, Amb a 1, 167 and shortly thereafter, a highly potent human IgG antibody (ie, BAB1) which could prevent allergic patients' IgE binding to the major birch pollen allergen Bet v 1 was isolated. 168 Of note, BAB1 also inhibited Bet v 1-induced basophil activation. In order to obtain IgG antibodies which are equivalent to IgE antibodies, a combinatorial library was constructed from peripheral blood mononuclear cells of a grass pollen allergic patient and for the first time human allergen-specific IgE Fabs were isolated. 169 One of these IgE Fabs which was specific for the major grass pollen allergen Phl p 2 could be converted into a complete recombinant human IgG antibody which blocked Phl p 2-induced basophil degranulation and hence had therapeutic potential. 170 In order to obtain recombinant human allergen-specific IgG which can already block the entry of allergens through the respiratory epithelium, bi-specific antibodies were created. In fact, it could be demonstrated that one can immobilize allergen-specific blocking IgG antibodies via ICAM1-specific IgG onto the respiratory epithelium and thus prevent allergen penetration 171 which opens the possibility for topical treatment with blocking antibodies.
The ultimate demonstration for the efficacy of passive immunization with allergen-specific recombinant antibodies for therapy of allergy was recently reported. Immunization with two monoclonal IgG 4 antibodies specific for the major cat allergen, Fel d 1, protected against cat allergy in a clinical study conducted in an exposure chamber. 36,37 The concept of passive immunization with allergen-specific recombinant IgG antibodies is intriguing and is certainly a possible approach for allergen sources which contain mainly one major allergen which can be blocked with one or few monoclonal antibodies. This approach will be particularly useful for seasonal allergies because one preseasonal immunization may be sufficient to protect the patient during seasonal allergen exposure. However, it will be very difficult, if not impossible to protect patients suffering from allergy caused by allergen sources containing several potent allergen components such as grass pollen allergy, house dust mite allergy, and peanut allergy to mention a few because this would require the production of multiple monoclonal antibodies. Conceptually, the demonstration that passive treatment with allergen-specific IgG is effective for AIT is important because it teaches that one requirement for successful AIT vaccine is the induction of high titers of allergen-specific blocking IgG antibodies. One can thus imagine for the future that recombinant active allergen-specific AIT approaches as well as passive immunization strategies will be available and can be given depending on the need of the patient, similar to active and passive immunization for prevention of infectious diseases.

| Cell-based therapy
Much has been learned about the robust induction of immunological tolerance from the field of transplantation immunology. [172][173][174][175] Everybody reduced. 175,178 However, the cell-based allergen-specific prevention approach is highly experimental but warrants further investigation in clinical trials once the major safety hurdles can be overcome.

| FUTURE NEEDS IN AIT
In Table 2  eases. This would speed up AIT vaccine development. Furthermore, molecular approaches will allow optimizing the vaccine antigen itself F I G U R E 3 Toward allergen-specific prevention of allergy. Using chips containing micro-arrayed allergen molecules (top), it is possible to identify the clinically relevant allergen molecules for a given population. According to these profiles IgE-and T-cell epitopes of these allergens can be mapped and used for the development of molecular approaches for allergen-specific prevention (middle part). Carrier-bound B-cell epitope-based peptides and recombinant hypoallergens may be used for prenatal vaccination of mothers to transmit blocking antibodies to the off-spring or for early postnatal vaccination (Bottom). The administration of allergen-specific blocking antibodies by passive immunization of mothers may also be considered for primary prevention. T-cell epitope-containing peptides may be used for early postnatal tolerance induction by molecular design for the desired purpose in addition to the optimization of adjuvants and forms as well as schedules of administration.

| Molecular AIT approaches for different allergic manifestations and new indications
AIT of moderate-to-severe asthma and AIT of food allergy are two indications which may benefit from molecular AIT strategies. Since severe side effects are a major disadvantage of OIT of food allergy and OIT can only be applied for digestion-resistant food allergens, SCIT with recombinant hypoallergenic derivatives may be considered. In fact, it has been shown that SCIT with a hypoallergenic derivative of the major fish allergen, parvalbumin, was safe and a protective IgG response could be elicited. [133][134][135] It is thus conceivable that one can develop SCIT approaches also for other forms of food allergy based on designing hypoallergenic food allergen molecules. Safety is a major issue for AIT of moderate-to-severe asthma. In fact, the approach of carrier-bound B-cell epitope-containing peptides which is a second-generation hypoallergenic approach characterized by a profound reduction in IgE-mediated allergenic activity and a reduction in T-cell-mediated side effects would seem particularly suitable for asthma treatment. 139 In fact, the grass pollen allergy vaccine, BM32, which was developed by this approach showed a good safety profile in clinical studies and beneficial effects on asthma symptoms were noted in the field study. The carrier-bound B-cell epitope-containing peptide approach is also unique among all AIT approaches as it does not boost IgE production. 137 It should therefore be useful for secondary preventive AIT approaches such as AIT to prevent the progression of rhinitis toward asthma and for the prevention of the progression of clinically silent IgE sensitization toward symptomatic allergy. However, it has also been shown recently that vaccination of nonallergic subjects with recombinant hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, was safe and induced allergen-specific IgG antibodies which prevented allergic patients IgE binding to Bet v 1. 188 In this study, the induction of allergenspecific IgE could be detected but due to the massive production of allergen-specific IgG no allergic sensitization occurred in the nonallergic subjects. Figure 3 provides an overview for allergen-specific approaches for primary prevention of allergy. A major prerequisite for molecular approaches of primary allergy prevention is the knowledge of the molecular sensitization profiles of the target population. The molecular sensitization profiles can be easily established using micro-arrayed allergen molecules. 7,189 According to the IgE reactivity profiles determined for a population, the most relevant allergen molecules can be selected according to prevalence of IgE reactivity and allergenic activity for the formulation of the preventive vaccines. It is clear that such a vaccine will not contain all molecules but data from birth cohort analyses have shown that one can define a reasonable number of important allergens. 124 For active vaccination SCIT approaches based on hypoallergenic allergen molecules such as recombinant hypoallergens and carrier-bound B-cell epitope-containing peptides may be considered as best approaches because these molecules exhibit low allergenic activity and induce allergen-specific IgG antibodies. Besides early postnatal vaccination one may also consider to build up high levels of allergen-specific IgG in females reaching peak levels in the end of pregnancy because evidence was provided that high levels of allergen-specific IgG transmitted from mothers to offsprings may protect against allergic sensitization. 190 In this context, also passive immunization with allergen-specific IgG could be possible but it will be difficult to make allergen-specific IgG antibodies for several different allergens. Another possibility for primary prevention of allergy could be early tolerance induction to suppress the development of allergen-specific adaptive immune responses. This could be achieved by the administration of hematopoietic stem cells, eventually cord blood cells, which are transfected with allergens or allergen-derived peptides. 174 Alternatively, oral tolerance induction by feeding allergens or allergen-derived T-cell epitope-containing peptides may be possible. 191,192 The latter approaches clearly involve ethical considerations and will require extensive research in experimental models, whereas the active vaccination approach of using hypoallergens could enter relatively soon clinical trials. These trials will have to show the safety and lack of allergic sensitization in nonallergic adults. Next, trials studying secondary prevention followed by trials investigating primary prevention in proof of principle studies using a frequently recognized allergen in a population with high sensitization rates toward this molecule can be conducted.

| Concluding remarks
AIT has a history of almost 110 years. It represents a therapeutic vaccination against allergy and has potential also for allergen-specific prevention. AIT is the only disease-modifying treatment for allergy and has several other important advantages over pharmacological treatments and biological therapies such as high clinical efficacy, long-lasting effects, and cost effectivity. However, AIT has also disadvantages such a side effects and cumbersome treatment schedules. Much has been learned about the mechanisms of AIT in numerous preclinical and clinical studies. With the availability of the disease-causing allergen molecules, a series of molecular AIT strategies has been developed which may revolutionize traditional allergen extract-based AIT to allow more patients to benefit from AIT and to develop preventive AIT forms.