Novel vaccines for allergen-specific immunotherapy

Purpose of review Allergen-specific immunotherapy (AIT) is a highly economic, effective and disease-modifying form of allergy treatment but requires accurate prescription and monitoring. New molecular approaches are currently under development to improve AIT by reducing treatment-related side effects, cumbersome protocols and patients’ compliance. We review the current advances regarding refined diagnosis for prescription and monitoring of AIT and the development of novel molecular vaccines for AIT. Finally, we discuss prophylactic application of AIT. Recent findings There is evidence that molecular allergy diagnosis not only assists in the prescription and monitoring of AIT but also allows a refined selection of patients to increase the likelihood of treatment success. New data regarding the effects of AIT treatment with traditional allergen extracts by alternative routes have become available. Experimental approaches for AIT, such as virus-like particles and cell-based treatments have been described. New results from clinical trials performed with recombinant hypoallergens and passive immunization with allergen-specific antibodies highlight the importance of allergen-specific IgG antibodies for the effect of AIT and indicate opportunities for preventive allergen-specific vaccination. Summary Molecular allergy diagnosis is useful for the prescription and monitoring of AIT and may improve the success of AIT. Results with molecular allergy vaccines and by passive immunization with allergen-specific IgG antibodies indicate the importance of allergen-specific IgG capable of blocking allergen recognition by IgE and IgE-mediated allergic inflammation as important mechanism for the success of AIT. New molecular vaccines may pave the road towards prophylactic allergen-specific vaccination.


INTRODUCTION
The burden of allergy is increasing globally and there is need for new therapies that improve the quality of life of allergic patients, reduce economic costs and are suitable for a precision approach [1-3,4 & ,5]. In 1911, Noon [6] was the first to show the benefit of AIT by administering the causative allergen to grass pollen allergic patients to treat the disease. AIT is associated with the induction of protective allergen-specific blocking IgG antibodies and cellular immune mechanisms [7,8 && ]. AIT is the only treatment that instructs the immune system of the patients for protection by therapeutic vaccination and can prevent the progression of the severity of allergic disease [9]. However, the use of crude allergens bears many inconveniences just as induction of side effects, lack of standardization resulting in poor immunogenicity, dose efficacy problems and limited efficacy. Moreover, low patients' compliance because of cumbersome treatment protocols in particular for sublingual immunotherapy (SLIT) [10,11 & ,12-14] is also considered key challenges for traditional AIT.
Advancements in molecular allergen characterization by DNA technology led to the development of new forms of AIT based on recombinant purified proteins, hypoallergenic derivatives and peptides [11 & ,15,16]. Moreover, biologics, such as monoclonal IgE antibodies and novel adjuvants have been considered for improving AIT in the last years [17].
This review highlights advances in the field of AIT placing an emphasis on molecular diagnosis for improving prescription and monitoring of AIT ( Fig. 1) as well as recent approaches for AIT ( Fig. 2 and Table 1). Furthermore, possible approaches for prophylactic vaccination against allergy are considered.

MOLECULAR ALLERGY DIAGNOSIS FOR PRESCRIPTION, MONITORING AND FOR ENHANCING TREATMENT SUCCESS OF ALLERGEN-SPECIFIC IMMUNOTHERAPY
On the basis of the fact that allergen sources contain allergen molecules, which are source-specific but also cross-reactive with unrelated sources, it has been proposed to use them for molecular diagnosis to better identify the genuinely sensitizing allergen sources for refined prescription of the AIT vaccines [18,19]. The advantage of defining the originally sensitizing allergen sources avoids that unnecessary administration of incorrect allergy vaccines is performed reducing possible side effects as well as treatment costs [20]. Initially this concept was misunderstood as it was thought that correct prescription would also increase efficacy of AIT but efficacy depends also on the quality of the AIT vaccine and the immune response of the treated patient to the vaccine. It was, therefore, also recommended to monitor the development of protective allergenspecific IgG antibodies in the course of the treatment, which by now is an accepted surrogate marker for the effects of AIT [8 && ,21]. The advantage of molecular testing for AIT-induced IgG is that it allows identifying if a patient develops protective IgG against those allergens against which the patient is sensitized [22 && ,23 && ]. Using allergen microarrays containing small amounts of immobilized allergens, it was found that the development of blocking IgG antibodies is reflected by a decrease of allergen-specific IgE binding through competition of IgG with IgE for the binding sites on the allergens. This decrease of the IgE binding also serves as surrogate marker for effective AIT [24][25][26].
In the meantime, molecular allergy diagnosis has been implemented as companion diagnostic tool for AIT [27 & ]. Multiallergen testing with allergen micro-arrays allows discriminating polysensitized from oligosensitized and monosensitized patients of whom the first may benefit more from symptomatic treatment whereas the latter may be more suitable for AIT ( Fig. 1).
Several reports show that clinicians can use molecular testing for refining AIT prescription and there is evidence that it reduces treatment costs [28][29][30] (Fig. 1). Very recently two studies have pointed out additional advantages of molecular diagnosis for AIT prescription. These studies demonstrated that patients who were sensitized to allergens, which were included in allergen extract-based vaccines had a better treatment success than patients who were sensitized also to other allergen molecules not included in the vaccines [

ALLERGEN EXTRACT-BASED ALLERGEN-SPECIFIC IMMUNOTHERAPY VACCINES ADMINISTERED BY ALTERNATIVE ROUTES
Although everybody is aware of the limitations of allergen extracts [10], several approaches are ongoing regarding AIT with allergen extracts that are administered via alternative routes ( Fig. 2 and Table 1). A recently published clinical phase III study performed as international, double-blind, placebocontrolled (DBPC) clinical trial investigated the clinical effects by assessing the total combined score in more than 1400 house dust mite allergic patients (Table 1). Patients were randomized into a placebo and a SLIT group receiving a tablet containing a mixture of Dermatophagoides pteronyssinus (Dp) and Dermatophagoides farinae (Df) extract for 1 year.
Precision medicine approach to allergy treatment by molecular allergy diagnosis. Molecular diagnosis identifies oligo-sensitized and mono-sensitized patients for AIT, guides prescription of AIT, allows refined selection of vaccines and monitoring of treatment effects. AIT, allergen-specific immunotherapy.
Active treatment achieved a relative least squares mean difference of 16.9% improvement of the total combined score in the tablet versus placebo group [33 && ]. The latter study reflects well the magnitude of effect of SLIT with tablets observed in earlier large AIT trials [34]. Like in other studies, there was a very low increase of allergen-specific IgG levels in the active group but a strong increase of allergen-specific IgE levels from median Dp-specific baseline levels of 14.81 kUA/l to median levels of Dp-specific IgE of 44.01 kUA/l was found. Treatment-related adverse events, mainly application site reactions occurred in more than 50% of the actively treated patients but were rare in the placebo group raising the question if SLIT studies can be effectively blinded.
Two recent intralymphatic immunotherapy (ILIT) studies showed that ILIT is clinically effective but the treatment-induced IgG antibody levels were not higher than in subcutaneous AIT (SCIT) studies and in one study no effective boost of antibody levels by the booster injection was observed [35 && ,36 && ] (Table 1). Therefore, it remains unclear whether ILIT has any advantages over SCIT.
Data from epicutaneous AIT (EPIT) studies performed with peanut allergen extracts provide evidence for clinical effects [37

,38
&& ] (Table 1) but these effects are modest, and a systematic review and metaanalysis [39] concludes that more studies are necessary to understand if this form of AIT is effective. Furthermore, the mechanisms of EPIT are not clear as it has been demonstrated that epicutaneous allergen administration induces no relevant increases of allergen-specific IgG antibodies but rather increases allergen-specific T-cell activation [40]. Viaskin is an epicutaneous immunotherapy formulation [41] currently in three different phase III clinical trials for pediatric patients and it is yet to receive approval for use and marketing. It has so far demonstrated some low induction of IgG 4 antibodies to certain peanut allergens [42 && ]. However, regulatory authorities have questioned its efficacy [43].
Oral immunotherapy (OIT) is mainly conducted with digestion-resistant food allergens [44] but does not seem to be applicable for respiratory allergy. Food OIT studies indicate that beneficial effects depend on the induction of allergen-specific IgG antibodies [45 && ] (Table 1). Palforzia from Aimmune Therapeutics (Brisbane, CA, USA) and an OIT formulation consisting of peanut protein increased in AIT approaches Overview of allergen-specific immunotherapy approaches including different application routes with allergenextract-based vaccines, molecular AIT approaches, nucleic acid-like, virus-like nanoparticle-based approaches and cellular forms of treatment. AIT, allergen-specific immunotherapy.
Novel vaccines for allergen-specific immunotherapy Akinfenwa et al.    67.2% the amount of peanut that patients could consume in a clinical trial [46]. However, it is still not without the risk of causing anaphylaxis and patients must still avoid a peanut diet but it is approved for use and marketing. However, a major disadvantage of OIT is that it induces severe side effects [47].

'MOLECULAR APPROACHES' WITH NATURAL ALLERGEN PREPARATIONS
Several early studies have shown that AIT with purified major allergens or chemically modified major allergen preparations is clinically effective [48][49][50]. In this context, the AIT study performed  (Fig. 2 and Table 1).
Another allergen extract-based approach has utilized a hydrolyzed allergen extract from Lolium perenne including peptides of a size between 1 and 10 kDa, which were administered without adjuvant. Initial studies indicated that the vaccine induces allergen-specific IgG responses [53 && ] and reduces allergic inflammation [54,55] (Table 1).
Despite the use of allergen peptides, which were thought to be hypoallergenic severe side effects were observed in the course of a randomized, multicenter DBPC trial [55]. There was evidence for clinical efficacy in a subset of patients but the phase III trial results did not meet the endpoints (https://www.businesswire.com/news/home/20191124005103/en/ ASIT-biotech-gp-ASIT%E2%84%A2-Phase-III-Trial-Grass).

RECOMBINANT HYPOALLERGENIC MOLECULES
The first generation of recombinant hypoallergenic molecules has been made to reduce IgE reactivity and allergenic activity (i.e. the ability of a molecule to induce IgE-dependent mast cells/basophil activation) of the molecules and at the same time to maintain allergen-specific T-cell epitopes [56] (Fig. 2). The first AIT trial, which was conducted with hypoallergenic derivatives of the major birch pollen allergen Bet v 1 indeed demonstrated that this approach may be effective and avoids immediate type side effects [57] but late phase side effects, were still observed [58]. Atopy patch test studies indicated that these late phase side effects are because of T-cell activation [59,60]. Accordingly, a refined second generation of recombinant hypoallergens was engineered to reduce the presence of allergen-specific T-cell epitopes but to maintain the ability of the derivatives to induce allergen-specific IgG antibodies [61] (Fig. 2). For this purpose, fusion proteins consisting of a nonallergenic carrier protein and nonallergenic peptides derived from the IgE epitopes of allergens were produced. A virus-derived carrier molecule provided additional T-cell help for the production of allergen-specific IgG production without activating allergen-specific T cells [62]. For the peptide-carrier fusion proteins made for treatment of grass pollen allergy (i.e. the BM32 vaccine) using the hepatitis B (HBV)-derived PreS protein as carrier it could be shown, that the PreScontaining vaccine induced also HBV-specific antibodies, which protected against HBV-infection in vitro [63]. In the meantime, the BM32 vaccine has been shown to be clinically effective in a multicenter DBPC phase IIb study [64] (Table 1) and is scheduled for phase III studies. The analysis of sera from a recent phase IIb study confirmed the robust induction of IgG antibodies against the domain of PreS, which is critical for the binding of the HBV virus to liver cells and demonstrated cross-reactivity with the most common HBV strains [65 && ] ( Table 1). Thus PreS-based allergy vaccines may be useful for vaccination against HBV.
Some additional results for BM32 were obtained in sub-studies. One sub-study compared the magnitude and specificity of allergen-specific IgG responses in patients treated with BM32 and a traditional, allergen extract-based registered SCIT [66 && ] (Table 1). It was found that much less injections of BM32 compared with the extract-based SCIT were needed to induce a comparable IgG response and the BM32 vaccination-focused IgG antibodies precisely to the IgE binding sites of the allergens. Another sub-study demonstrated that vaccination with BM32 induced continuously growing allergenspecific IgG 4 responses and did not activate allergenspecific T-cell responses [67 && ] ( Table 1). A few preclinical results describing recombinant vaccine constructs may also be mentioned. For example, basophil activation experiments demonstrated that a hybrid consisting of major allergens from two different aeroallergen sources, Bet v 1 and Phl p 5 induced a much reduced activation, compared with the monomeric forms of the proteins [68]. Similarly, fusion proteins consisting of four antigenic regions from the major allergens of the mite species D. pteronyssinus showed a significantly reduced IgE reactivity, compared with the individual proteins (Der p 1, Der p 2 and Der p 7) [69]. Mice immunized with the HDM hybrid protein generated blocking IgG antibodies, thus showing a possibility for a novel HDM vaccine [69].

SYNTHETIC HYPOALLERGENIC PEPTIDES
Allergen-derived synthetic peptides containing Tcell epitopes and lacking IgE reactivity have been considered for AIT for almost 30 years. This approach was thought to induce T-cell tolerance with the hope that it would reduce allergen-specific IgE production and induce protective allergen-specific IgG responses. The AIT studies performed in experimental animal models and in clinical trials in humans provide indeed evidence that peptide-based AIT may induce signs of immunological tolerance ( Fig. 2 and Table 1). In a recent study, a downregulation of a chemokine receptor, chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTh2) in patients that had received a Fel d 1 peptide vaccine (Cat-PAD) was reported [70 & ]. This mechanistic sub-study was part of a phase III trial, which showed no effects of treatment over placebo [70 & ]. The AIT trials performed with T-cell epitope-containing peptides could never show unambiguous effects of treatment on the production of allergen-specific IgE or IgG whenever performed with short peptides.
An induction of allergen-specific IgG antibodies was only observed with longer, immunogenic peptides as reported in a phase IIb trial performed with contiguous overlapping peptides from Bet v 1 [71 & ] ( Fig. 2 and Table 1). In one of the studies, a significant clinical improvement of more than 20% compared with the placebo group was observed. This was associated with increased Bet v 1-specific IgG 4 antibodies up to two pollen seasons after treatment [71 & ] ( Table 1). It thus seems that AIT with allergenderived peptides requires the induction of allergen-specific IgG to achieve clinical benefit and that such an effect can only be obtained with long and immunogenic peptides.
Although the induction of T-cell tolerance by peptide AIT remains challenging, one may consider this approach for preventive tolerance induction. In this context, approaches, such as prophylactic oral tolerance induction with T-cell epitope-containing peptides have been reconsidered for allergen-specific prevention [72]. A recent study performed in murine model of fish allergy showed that prophylactic feeding with the major fish allergen parvalbumin induced tolerance point to a T-cell-mediated, tolerogenic effect [73 & ]. However, further studies will be necessary to investigate if such an effect can also be obtained with synthetic allergen-derived peptides. Another recent study prepares the ground for such experiments by defining a panel of synthetic hypoallergenic peptides covering the major house dust mite allergens, Der p 1, Der p 2, Der p 5, Der p 7, Der p 21 and Der p 23 [74].

IMMUNOTHERAPY BY PASSIVE IMMUNIZATION
In 1935, it was shown by Cooke et al. [75] that serum from AIT-treated patients containing allergen-specific IgG protects against allergic skin inflammation. This study confirmed the earlier work by Dunbar in 1903 [76], which demonstrated that pollen allergenspecific antisera neutralized the allergenic activity of pollen allergens. These historic studies indicated that it may be possible to use allergen-specific IgG antibodies, which block allergic patients IgE binding to allergens for treatment of allergy by passive immunization.
The great potential of the passive immunization approach was recently shown in a clinical study performed in cat allergic patients who were passively vaccinated with monoclonal IgG antibodies against the major cat allergen, Fel d 1 [77] (Fig. 2 and Table 1). The injection of antibodies improved the allergic symptoms of the allergic patients, caused few side effects and a correlation between clinical symptoms and the IgG/IgE ratio was shown. The passive immunization approach is also supported by experimental animal studies.
It was shown in a murine model of fish allergy that passive immunization with IgG antibodies specific for a hypoallergenic mutant of the major fish allergen, parvalbumin reduced allergic symptoms [78]. In another study performed in a model of peanut allergy, transfer of allergen-specific IgG antibodies led to a decreased response in local and systemic reactions after allergen challenge with peanut extract [79 & ]. Major challenges for such approach are the high costs required for the production of large quantities of antibodies and the fact, that for certain allergen sources containing more than one major allergen, cocktails of several monoclonal antibodies will be needed to achieve sufficient blocking of IgE binding to each of the allergens. Moreover, certain allergens contain multiple IgE epitopes which could not be neutralized even with several different monoclonal antibodies [80].

NUCLEIC ACID VACCINES FOR ALLERGEN-SPECIFIC IMMUNOTHERAPY
Nucleic acid vaccines are based on the application of DNA or RNA to produce the antigen in transfected cells of the host instead of immunizing with the antigen. This approach was already described in 1992 [81]. In 1993, it was demonstrated that the injection with DNA encoding a viral protein induced protection by boosting both T cell and antibody responses [82]. The approach was then tested for allergy in animal models and it was shown that it could mitigate an allergen-specific immune response [83,84] (Fig. 2). However, the technology was tested only recently in clinical trials. A phase 1A and 1B study was performed with the Japanese cedar pollen DNA vaccine encoding the major allergen Cry j 2. The study was conducted in a region where no Japanese cedar trees were growing, therefore, natural exposure could not be determined. Additionally, the study did not include a mock control group and the treated patients experienced 88 different adverse events in total [85] (Table 1).
A drawback of genetic vaccination is the weak induction of blocking IgG antibodies. Therefore, several methods regarding the route of delivery have been studied, such as gene gun vaccination or electroporation [86].
Other possible problems with nucleic acid-based vaccination may result from the integration of the vaccine into the genome, the long-term effects of the plasmid DNA, the development of anti-DNA antibodies and the long-term expression of the allergen encoded, which could lead to the development of adverse events or even more severe inflammatory diseases [87].

VIRUS-LIKE NANOPARTICLES
Using a model of mugwort pollen allergy [88], it was demonstrated that inclusion of the complete major mugwort pollen allergen, Art v 1 within VNPs resulted in a nonanaphylactogenic vaccine when tested in basophil activation tests [89 & ] ( Fig. 2 (Table 1). In a preclinical study with a vaccine candidate against peanut allergy where Ara h 1 or Ara h 2 were coated to the cucumber mosaic virus-derived particle (CMV), positive results were obtained. The vaccine-induced protective responses in mice [79 & ] (Table 1). However, the VNP approach has so far been only tested in preclinical models, the vaccines may be difficult to produce under Good Manufacturing Practice (GMP) conditions suitable for clinical trials, and accordingly there is so far no experience in clinical AIT studies.

CELL-BASED THERAPY
Different cell-based approaches for prevention and treatment of allergy are emerging ( Fig. 2 and Table 1). One approach is based on the prophylactic administration of allergen-expressing leukocytes into new-born recipients with the goal to induce allergenspecific immune tolerance at the cellular and humoral levels. Results from experimental animal studies provide evidence that such a treatment can prevent the development of allergic sensitization [91]. Another different approach utilizes chimeric antigen receptor (CAR) T cells for targeting cells of the allergic immune response. CAR T cells were originally made for cancer immunotherapy and are engineered to express an immunoreceptor, which can specifically recognize certain targets on cells and are linked with T-cell activating functions. Several applications for the treatment of allergy and asthma by CAR T cells have been discussed [92 & ]. For example, it has been suggested to engineer CAR T cells to target cells expressing membrane-bound IgE (mIgE) similar as has been done with a monoclonal antibody-based therapy earlier [93]. Although the CAR T-cell approach is currently considered to be used as a nonallergen-specific form of treatment, it may be envisaged that this approach could be used to target also allergen-specific T cells or B cells producing allergen-specific IgE via the variable regions of the T-cell receptors or membrane IgE but the polyclonality of allergen-specific T-cell receptors will be a major hurdle for an allergen-specific approach. In addition, CAR T-cell approaches may cause side effects, such as inflammation, neurotoxicity, anaphylaxis, immunological rejection, cellular injury, insertional oncogenesis and cytokine storm (CRS) [92 & ].

PROPHYLACTIC ALLERGEN-SPECIFIC VACCINATION
There is growing evidence that prophylactic allergen-specific vaccination may be a possibility for preventing allergies. Data obtained by studying allergic sensitization at the molecular level in birth cohorts suggest several windows of opportunity for interrupting or preventing the process of allergic sensitization by tolerance induction or allergen-specific vaccination [94] (Table 1). A recent perspective article has highlighted the possible steps for developing and evaluating prophylactic vaccination concepts and suggests that recombinant hypoallergenic molecules may be best suited for preventive vaccination [95 & ].
In this context, a first controlled vaccination study performed with hypoallergenic recombinant allergen derivatives of the major birch pollen allergen, Bet v 1 in nonallergic individuals should be mentioned (Table 1). This study [96 && ] demonstrated that vaccination with the hypoallergenic Bet v 1 derivatives could induce normal Bet v 1-specific IgG responses in nonallergic individuals and that these IgG antibodies blocked allergic patients' IgE recognition of Bet v 1. Even more important, this study showed that vaccination with the hypoallergenic derivatives did not induce allergic sensitization in the nonallergic individuals [96 && ]. The study may thus be considered as a first step towards prophylactic allergen-specific vaccination.

CONCLUSION
AIT is an effective, cost-effective and disease-modifying treatment for allergy with long-lasting effects.
It is ideally suited for a precision medicine approach for managing allergic diseases. Molecular allergy diagnosis is useful for prescription, monitoring and even for prediction of treatment success and has become an integral part of modern allergy diagnosis. Modern forms of molecular vaccines are urgently needed to improve AIT, to render it useful for broad application to respiratory, food and other forms of allergy and for prophylactic use. Certain molecular AIT concepts have been successful in clinical trials and need to be carried forward vigorously to catch up with advances made in molecular allergy diagnosis to provide best practice precision medicine for allergic patients.