Vaccine development for allergen-specific immunotherapy based on recombinant allergens and synthetic allergen peptides: Lessons from the past and novel mechanisms of action for the future
Authors: Rudolf Valenta, Raffaela Campana, Margit Focke-Tejkl, Verena Niederberger
Journal: Journal of Allergy and Clinical Immunology (2016)

In the past, the development of more effective, safe, convenient, broadly applicable, and easy to manufacture vaccines for allergen-specific immunotherapy (AIT) has been limited by the poor quality of natural allergen extracts. Progress made in the field of molecular allergen characterization has now made it possible to produce defined vaccines for AIT and eventually for preventive allergy vaccination based on recombinant DNA technology and synthetic peptide chemistry. Here we review the characteristics of recombinant and synthetic allergy vaccines that have reached clinical evaluation and discuss how molecular vaccine approaches can make AIT more safe and effective and thus more convenient. Furthermore, we discuss how new technologies can facilitate the reproducible manufacturing of vaccines of pharmaceutical grade for inhalant, food, and venom allergens. Allergy vaccines in clinical trials based on recombinant allergens, recombinant allergen derivatives, and synthetic peptides allow us to target selectively different immune mechanisms, and certain of those show features that might make them applicable not only for therapeutic but also for prophylactic vaccination. In the past, the development of more effective, safe, convenient, broadly applicable, and easy to manufacture vaccines for allergen-specific immunotherapy (AIT) has been limited by the poor quality of natural allergen extracts. Progress made in the field of molecular allergen characterization has now made it possible to produce defined vaccines for AIT and eventually for preventive allergy vaccination based on recombinant DNA technology and synthetic peptide chemistry. Here we review the characteristics of recombinant and synthetic allergy vaccines that have reached clinical evaluation and discuss how molecular vaccine approaches can make AIT more safe and effective and thus more convenient. Furthermore, we discuss how new technologies can facilitate the reproducible manufacturing of vaccines of pharmaceutical grade for inhalant, food, and venom allergens. Allergy vaccines in clinical trials based on recombinant allergens, recombinant allergen derivatives, and synthetic peptides allow us to target selectively different immune mechanisms, and certain of those show features that might make them applicable not only for therapeutic but also for prophylactic vaccination. Allergen-specific immunotherapy (AIT) was reported first by Leonard Noon in 1911.1Noon L. Prophylactic inoculation against hay fever.Lancet. 1911; 1: 1572-1573Abstract Scopus (1148) Google Scholar Noon injected grass pollen extract into allergic patients and, despite the occurrence of side effects, observed clinical improvement for almost 1 year in the treated patients. In his article Noon quotes earlier work by William Dunbar,2Dunbar W.P. Zur Frage betreffend die Ätiologie und spezifische Therapie des Heufiebers. Berliner klin.Wochenschr. 1903; : 24-26Google Scholar who had shown that anti-sera raised against pollen allergen extract could neutralize allergen-induced conjunctival inflammation. The early work of Dunbar had already indicated that a major effect of AIT was caused by induction of allergen-specific blocking antibodies. In 1935, more than 40 years before the identification of IgE antibodies, Cooke et al3Cooke R.A. Barnard J.H. Hebald S. Stull A. Serological evidence of immunity coexisting sensitization in a type of human allergy (hay fever).J Exp Med. 1935; 62: 733-750Crossref PubMed Scopus (259) Google Scholar reported a series of elegant experiments showing that allergen-specific IgG antibodies induced by AIT can suppress allergen-induced skin inflammation. Since then, the importance of allergen-specific IgG antibodies that compete with IgE for binding to the allergens has been demonstrated by numerous studies as a major mechanism of the mode of action of AIT,4Larche M. Akdis C.A. Valenta R. Immunological mechanisms of allergen-specific immunotherapy.Nat Rev Immunol. 2006; 6: 761-771Crossref PubMed Scopus (645) Google Scholar and therefore one might consider AIT and in particular the traditional form of subcutaneous AIT as a therapeutic vaccine.5Bousquet J. Lockey R. Malling H.J. Allergen immunotherapy: therapeutic vaccines for allergic diseases. A WHO position paper.J Allergy Clin Immunol. 1998; 102: 558-562Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar There are several important features that suggest that AIT has many advantages over symptomatic treatment with anti-inflammatory drugs and biologics when applied as recommended according to guidelines.6Jutel M. Agache I. Bonini S. Burks A.W. Calderon M. Canonica W. et al.International consensus on allergy immunotherapy.J Allergy Clin Immunol. 2015; 136: 556-568Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar First of all, AIT functions in an allergen-specific and thus causative manner as a therapeutic vaccine. It uses the immune system of the patient to establish a counterimmune response, antagonizing the allergic immune response by vaccination with the disease-causing allergens or derivatives thereof. Therefore, as with other vaccines, allergy vaccines can be relatively easily produced, and the costs of AIT are low, in particular when compared with those of treatment with biologic agents, such as anti-cytokine antibodies.7Cox L. Calderón M. Pfaar O. Subcutaneous allergen immunotherapy for allergic disease: examining efficacy, safety and cost-effectiveness of current and novel formulations.Immunotherapy. 2012; 4: 601-616Crossref PubMed Scopus (44) Google Scholar Unlike anti-inflammatory treatment, AIT can stop the progression of mild forms (ie, rhinitis) of allergy toward severe forms (ie, asthma) and thus modifies the natural course of disease.8Jacobsen L. Niggemann B. Dreborg S. Ferdousi H.A. Halken S. Host A. et al.Specific immunotherapy has long-term preventive effect of seasonal and perennial asthma: 10-year follow-up on the PAT study.Allergy. 2007; 62: 943-948Crossref PubMed Scopus (821) Google Scholar, 9Valovirta E. Effect of AIT in children including potential to prevent the development of asthma.Allergy. 2011; 66: 53-54Crossref PubMed Scopus (14) Google Scholar Furthermore, AIT has long-lasting effects, even after discontinuation of treatment, which cannot be achieved with symptomatic treatment.10Durham S.R. Walker S.M. Varga E.M. Jacobson M.R. O'Brien F. Noble W. et al.Long-term clinical efficacy of grass-pollen immunotherapy.N Engl J Med. 1999; 341: 468-475Crossref PubMed Scopus (1274) Google Scholar The achievement of long-term "clinical tolerance" can be achieved through induction of long-lived B cells or plasma cells secreting high-affinity antibodies11James L.K. Shamji M.H. Walker S.M. Wilson D. Wachholz P.A. Francis J.N. et al.Long-term tolerance after allergen immunotherapy is accompanied by selective persistence of blocking antibodies.J Allergy Clin Immunol. 2011; 127: 509-516Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar and/or through a reduction in boosts of allergen-specific IgE, which occurs after natural allergen contact.12Mothes N. Heinzkill M. Drachenberg K.J. Sperr W.R. Krauth M.T. Majlesi Y. et al.Allergen-specific immunotherapy with monophosphoryl lipid A-adjuvanted vaccine: reduced seasonally boosted immunoglobulin E production and inhibition of basophil histamine release by therapy-induced blocking antibodies.Clin Exp Allergy. 2003; 33: 1198-1208Crossref PubMed Scopus (245) Google Scholar, 13Niederberger V. Horak F. Vrtala S. Spitzauer S. Krauth M.T. Valent P. et al.Vaccination with genetically engineered allergens prevents progression of allergic disease.Proc Natl Acad Sci U S A. 2004; 101: 14677-14682Crossref PubMed Scopus (336) Google Scholar, 14Creticos P.S. Schroeder J.T. Hamilton R.G. Balcer-Whaley S.L. Khattignavong A.P. Lindblad R. et al.Immunotherapy with a ragweed-toll-like receptor 9 agonist vaccine for allergic rhinitis.N Engl J Med. 2006; 355: 1445-1455Crossref PubMed Scopus (509) Google Scholar Diagnosis of the disease-causing allergens and monitoring of treatment have been greatly facilitated through the availability of molecular allergy diagnosis, also termed component-resolved allergy diagnosis.15Hiller R. Laffer S. Harwanegg C. Huber M. Schmidt W.M. Twardosz A. et al.Microarrayed allergen molecules: diagnostic gatekeepers for allergy treatment.FASEB J. 2002; 16: 414-416Crossref PubMed Scopus (405) Google Scholar, 16Lupinek C. Wollmann E. Baar A. Banerjee S. Breiteneder H. Broecker B.M. et al.Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.Methods. 2014; 66: 106-119Crossref PubMed Scopus (185) Google Scholar, 17Canonica G.W. Ansotequi I.L. Pawankar R. Schmid-Grendelmeier P. van Hage M. Baena-Cagnani C.E. et al.A WAO-ARIA-GA2LEN consensus document on molecular-based allergy diagnostics.World Allergy Organ J. 2013; 6: 17Crossref PubMed Scopus (330) Google Scholar, 18Sastre J. Landivar M.E. Ruiz-García M. Andregnette-Rosigno M.V. Mahillo I. How molecular diagnosis can change allergen-specific immunotherapy prescription in a complex pollen area.Allergy. 2012; 67: 709-711Crossref PubMed Scopus (175) Google Scholar For example, microarrayed allergen molecules allow detection of IgE reactivities and treatment-induced IgG antibody responses toward a comprehensive set of allergens.16Lupinek C. Wollmann E. Baar A. Banerjee S. Breiteneder H. Broecker B.M. et al.Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.Methods. 2014; 66: 106-119Crossref PubMed Scopus (185) Google Scholar Thus tools are available for more precise prescription of allergy vaccines and for controlling the effects of the vaccine.16Lupinek C. Wollmann E. Baar A. Banerjee S. Breiteneder H. Broecker B.M. et al.Advances in allergen-microarray technology for diagnosis and monitoring of allergy: the MeDALL allergen-chip.Methods. 2014; 66: 106-119Crossref PubMed Scopus (185) Google Scholar, 19Wollmann E. Lupinek C. Kundi M. Selb R. Niederberger V. Valenta R. Reduction in allergen-specific IgE binding as measured by microarray: a possible surrogate marker for effects of specific-immunotherapy.J Allergy Clin Immunol. 2015; 136: 806-809Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar However, there are several important bottlenecks (Fig 1) that limit the broad applicability of AIT for allergy treatment. In this review we will discuss how these bottlenecks have been addressed in the past with traditional technologies and how modern technologies of molecular treatment might lead to a breakthrough of AIT not only for global allergy treatment but also ultimately for allergy prevention. Fig 1 provides an overview of areas in which improvement of AIT is needed. Some of these areas (safety, efficacy, and convenience) are closely connected to each other. A major problem in AIT is that administration of allergens can induce side effects in patients, which, in the worst-case scenario, can lead to anaphylactic shock and death.20Winther L. Arnved J. Malling H.J. Nolte H. Mosbech H. Side-effects of allergen-specific immunotherapy: a prospective multi-centre study.Clin Exp Allergy. 2006; 36: 254-260Crossref PubMed Scopus (122) Google Scholar Side effects can be classified as immediate side effects, which are caused by allergen-induced cross-linking of mast cell– and basophil-bound IgE antibodies. These side effects occur within 30 minutes after administration of the vaccine and, when induced systemically, can give rise to life-threatening anaphylactic shock. Systemic activation of mast cells and basophils occurs mainly when relevant doses of IgE-reactive allergens are distributed systemically in the body. A reduction in the risk of immediate systemic side effects can be achieved by keeping allergens locally bound at the application site, such as through the use of certain adjuvants, such as aluminum hydroxide, which has been introduced already in 1935 and led to a profound reduction of severe systemic side effects (Fig 2).21Sledge R.F. Treatment of hay-fever with alum-precipitated pollen extract.U S Naval Bull. 1938; 36: 18-29Google Scholar Another way to reduce side effects has been reduction of the IgE reactivity of allergen extracts, which in the past has been achieved by chemical modification, such as denaturation with aldehydes.22Marsh D.G. Lichtenstein L.M. Campbell D.H. Studies on "allergoids" prepared from naturally occurring allergens. I. Assay of allergenicity and antigenicity of formalinized rye group I component.Immunology. 1970; 18: 705-722PubMed Google Scholar Such modified allergen extracts that exhibit reduced IgE reactivity are termed "allergoids" (Fig 2). Interestingly, even strong reduction of IgE reactivity cannot eliminate side effects because late-phase side effects can occur even in the absence of IgE reactivity caused by the presence of undestroyed T-cell epitopes. In a classical study it has been shown that even non–IgE-reactive T-cell epitope–containing allergen peptides can induce systemic late-phase side effects that occur after hours and are caused by IgE-independent activation of allergen-specific T cells.23Haselden B.M. Kay A.B. Larché M. Immunoglobulin E-independent major histocompatibility complex-restricted T cell peptide epitope-induced late asthmatic reactions.J Exp Med. 1999; 189: 1885-1894Crossref PubMed Scopus (314) Google Scholar Late-phase side effects have been also reported for AIT with allergoids made from natural allergen extracts.24Norman P.S. Lichtenstein L.M. Marsh D.G. Studies on allergoids from naturally occurring allergens. IV. Efficacy and safety of long-term allergoid treatment of ragweed hay fever.J Allergy Clin Immunol. 1981; 68: 460-470Abstract Full Text PDF PubMed Scopus (74) Google Scholar, 25Bousquet J. Hejjaouim A. Soussana M. Michel F.B. Double-blind, placebo-controlled immunotherapy with mixed grass-pollen allergoids. IV. Comparison of safety and efficacy of two dosages of a high-molecular-weight allergoid.J Allergy Clin Immunol. 1990; 85: 490-497Abstract Full Text PDF PubMed Scopus (115) Google Scholar Because the IgE reactivity of allergoids is usually strongly reduced, these late-phase side effects can be also mediated by activation of allergen-specific T cells because allergoids retain their ability to stimulate T cells.26Kahlert H. Grage-Griebenow E. Stüwe H.T. Cromwell O. Fiebig H. T cell reactivity with allergoids: influence of the type of APC.J Immunol. 2000; 165: 1807-1815Crossref PubMed Scopus (46) Google Scholar Another possibility to reduce the risk of side effects is to begin treatment with very low doses and to continuously increase the dose until a therapeutically effective maintenance dose has been reached. As a result of the need for updosing, AIT requires multiple administrations, which make the treatment inconvenient. Therefore alternative routes of administration, such as sublingual, oral, and epicutaneous application, were developed, but these treatments also require frequent administration and are inconvenient.27von Moos S. Johansen P. Tay F. Graf N. Kündig T.M. Senti G. Comparing safety of abrasion and tape-stripping as skin preparation in allergen-specific epicutaneous immunotherapy.J Allergy Clin Immunol. 2014; 134: 965-967Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Spina L. Weisskopf M. von Moos S. Graf N. Kündig T.M. Senti G. Comparison of microneedles and adhesive-tape stripping in skin preparation for epicutaneous allergen delivery.Int Arch Allergy Immunol. 2015; 167: 103-109Crossref PubMed Scopus (19) Google Scholar, 29Sato S. Yanagida N. Ogura K. Imai T. Utsunomiya T. Iikura K. et al.Clinical studies in oral allergen-specific immunotherapy: differences among allergens.Int Arch Allergy Immunol. 2014; 164: 1-9Crossref PubMed Scopus (42) Google Scholar, 30Durham S.R. Yang W.H. Pedersen M.R. Johansen N. Rak S. Sublingual immunotherapy with once-daily grass allergen tablets: a randomized controlled trial in seasonal allergic rhinoconjunctivitis.J Allergy Clin Immunol. 2006; 117: 802-809Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar For example, sublingual treatment requires daily administration, and therefore it is not surprising that the compliance of patients receiving AIT is low and particularly low for sublingual AIT.31Keil M.A. Röder E. Gerth van Wijk R. Al M.J. Hop W.C. Rutten-van Mölken M.P. Real-life compliance and persistence among users of subcutaneous and sublingual allergen immunotherapy.J Allergy Clin Immunol. 2013; 132: 353-360Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar Another area of AIT in which improvement is needed is clinical efficacy. Very often it is not possible to reach and maintain the therapeutically active dose in patients because of side effects. Natural allergen extracts contain a variety of allergens with widely varying potency in different quantities. Several studies have shown that natural allergen extracts from different manufacturers contain widely varying contents of allergens and that often important allergens are lacking.32Focke M. Marth K. Flicker S. Valenta R. Heterogeneity of commercial timothy grass pollen extracts.Clin Exp Allergy. 2008; 38: 1400-1408Crossref PubMed Scopus (110) Google Scholar, 33Casset A. Mari A. Purohit A. Resch I. Weghofer M. Ferrara R. et al.Varying allergen composition and content affects the in vivo allergenic activity of commercial Dermatophagoides pteronyssinus extracts.Int Arch Allergy Immunol. 2012; 159: 253-262Crossref PubMed Scopus (128) Google Scholar, 34Casset A. Valenta R. Vrtala S. Allergen content and allergenic activity of house dust mite extracts.Int Arch Allergy Immunol. 2013; 161: 287-288Crossref PubMed Scopus (13) Google Scholar It is also known that individual allergens possess varying immunogenicity (ie, ability to induce IgG responses). For example, certain grass pollen allergens induce high IgG responses (eg, Phl p 5), whereas others induce only low or no IgG responses (eg, Phl p 2 and Phl p 1).12Mothes N. Heinzkill M. Drachenberg K.J. Sperr W.R. Krauth M.T. Majlesi Y. et al.Allergen-specific immunotherapy with monophosphoryl lipid A-adjuvanted vaccine: reduced seasonally boosted immunoglobulin E production and inhibition of basophil histamine release by therapy-induced blocking antibodies.Clin Exp Allergy. 2003; 33: 1198-1208Crossref PubMed Scopus (245) Google Scholar, 35Linhart B. Jahn-Schmid B. Verdino P. Keller W. Ebner C. Kraft D. et al.Combination vaccines for the treatment of grass pollen allergy consisting of genetically engineered hybrid molecules with increased immunogenicity.FASEB J. 2002; 16: 1301-1303PubMed Google Scholar, 36Gadermaier E. Staikuniene J. Scheiblhofer S. Thalhamer J. Kundi M. Westritschnig K. et al.Recombinant allergen-based monitoring of antibody responses during injection grass pollen immunotherapy and after 5 years of discontinuation.Allergy. 2011; 66: 1174-1182Crossref PubMed Scopus (29) Google Scholar Accordingly, allergen extract–based forms of AIT can induce only partial protection. This brings us to the major bottleneck for the further development of AIT, which is related to the use of natural allergen extracts as a source for manufacturing the vaccines. The huge variations regarding amounts, potencies, and immunogenicity of individual allergen molecules in natural allergen extracts cannot be controlled or even manipulated by pharmaceutical production processes. Also, modern technologies, such as proteomics tools or mass spectrometry, do not allow the adequate quantification of immunogenic allergens or allergen derivatives in extracts.34Casset A. Valenta R. Vrtala S. Allergen content and allergenic activity of house dust mite extracts.Int Arch Allergy Immunol. 2013; 161: 287-288Crossref PubMed Scopus (13) Google Scholar Therefore one cannot expect any further substantial improvement of AIT based on traditional allergen extract–based forms of treatment.37Focke M. Swoboda I. Marth K. Valenta R. Developments in allergen-specific immunotherapy: from allergen extracts to allergy vaccines bypassing allergen-specific immunoglobulin E and T cell reactivity.Clin Exp Allergy. 2010; 40: 385-397Crossref PubMed Scopus (96) Google Scholar Requirements for innovative forms of AIT are summarized in Fig 1 and include high safety. The vaccine should have no anaphylactic potential and induce as few systemic late-phase side effects as possible. AIT vaccines should be effective and induce suppression of clinical symptoms early after the start of the treatment and should have sustained effects, even after discontinuation of the treatment. It should be possible to achieve the therapeutic effect with few doses of application to increase patient compliance. Manufacturing of the vaccine must be reproducible and follow the pharmaceutical requirements set for vaccines and should be possible at low costs. The technology used for the production of the vaccine and the resulting vaccines should be applicable for all allergen sources, including respiratory, food, and venom allergens. Finally and importantly, it has become clear that one of the big advantages of AIT is that it can prevent the progression of mild to severe forms of allergy. It has been shown that AIT can prevent the progression of rhinitis to asthma when given in children.8Jacobsen L. Niggemann B. Dreborg S. Ferdousi H.A. Halken S. Host A. et al.Specific immunotherapy has long-term preventive effect of seasonal and perennial asthma: 10-year follow-up on the PAT study.Allergy. 2007; 62: 943-948Crossref PubMed Scopus (821) Google Scholar, 9Valovirta E. Effect of AIT in children including potential to prevent the development of asthma.Allergy. 2011; 66: 53-54Crossref PubMed Scopus (14) Google Scholar Through analysis of the evolution of IgE responses in early childhood with microarrayed allergen molecules in birth cohorts, it seems to become possible to predict the transition of silent and asymptomatic IgE sensitization toward the development of allergic symptoms.38Bousquet J. Anto J. Sunyer J. Nieuwenhuijsen M. Vrijheid M. Keil T. et al.Pooling birth cohorts in allergy and asthma: European Union-funded initiatives—a MeDALL, CHICOS, ENRIECO, and GA2LEN joint paper.Int Arch Allergy Immunol. 2013; 161: 1-10Crossref PubMed Scopus (52) Google Scholar, 39Westman M. Lupinek C. Bousquet J. Andersson N. Pahr S. 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Prosperi M.C. et al.Evolution pathways of IgE responses to grass and mite allergens throughout childhood.J Allergy Clin Immunol. 2015; 136: 1645-1652.e8Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Accordingly, one might consider applying AIT already to children who are sensitized but do not yet have symptoms to prevent the development of allergic symptoms or even as early intervention to prevent the development of allergic sensitization.42Valenta R. Campana R. Marth K. van Hage M. Allergen-specific immunotherapy: from therapeutic vaccines to prophylactic approaches.J Intern Med. 2012; 272: 144-157Crossref PubMed Scopus (93) Google Scholar In fact, we are beginning to see the first studies that make an attempt toward prophylactic AIT.43Szépfalusi Z. Bannert C. Ronceray L. Mayer E. Hassler M. Wissmann E. et al.Preventive sublingual immunotherapy in preschool children: first evidence for safety and pro-tolerogenic effects.J Allergy Clin Immunol. 2014; 25: 788-795Google Scholar, 44Zolkipli Z. Roberts G. Cornelius V. Clayton B. Pearson S. Michaelis L. et al.Randomized controlled trial of primary prevention of atopy using house dust mite allergen oral immunotherapy in early childhood.J Allergy Clin Immunol. 2015; 136: 1541-1547.e11Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 45Du Toit G. Roberts G. Sayre P.H. Bahnson H.T. Radulovic S. Santos A.F. et al.Randomized trial of peanut consumption in infants at risk for peanut allergy.N Engl J Med. 2015; 372: 803-813Crossref PubMed Scopus (1375) Google Scholar However, it will be necessary to have innovative AIT technologies that selectively induce protective IgG responses and/or tolerance without inducing and/or boosting IgE responses for preventive AIT. In this article we focus on defined molecular approaches for improvement of specific immunotherapy, which are based on purified recombinant allergens, recombinant allergen derivatives, and allergen-derived peptides. Problems associated with the bad quality of natural allergen extracts can be overcome by the use of purified recombinant allergen molecules, which can be produced in reproducible quality according to pharmaceutical requirements. Fusion of several allergens or of hypoallergenic allergen derivatives in the form of hybrid molecules allows for combining several allergens within one molecule and increasing the immunogenicity of the individual components.35Linhart B. Jahn-Schmid B. Verdino P. Keller W. Ebner C. Kraft D. et al.Combination vaccines for the treatment of grass pollen allergy consisting of genetically engineered hybrid molecules with increased immunogenicity.FASEB J. 2002; 16: 1301-1303PubMed Google Scholar, 46Linhart B. Hartl A. Jahn-Schmid B. Verdino P. Keller W. Krauth M.T. et al.A hybrid molecule resembling the epitope spectrum of grass pollen for allergy vaccination.J Allergy Clin Immunol. 2005; 115: 1010-1016Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar In a classical study Pauli et al47Pauli G. Larsen T.H. Rak S. Horak F. Pastorello E. Valenta R. et al.Efficacy of recombinant birch pollen vaccine for the treatment of birch-allergic rhinoconjunctivitis.J Allergy Clin Immunol. 2008; 122: 951-960Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar have demonstrated that rBet v 1 was equally effective as birch pollen extract for subcutaneous AIT of patients with birch pollen allergy. However, rBet v 1 retains the allergenic properties of nBet v 1, and therefore patients had to be treated with a rather inconvenient updosing schedule and monthly maintenance injections in this trial. To increase the safety of AIT based on rBet v 1, tablet formulations of rBet v 1 for sublingual treatment are currently being developed that use pharmaceutical grade recombinant allergen,48Nony E. Bouley J. Le Mignon M. Lemoine P. Jain K. Horiot S. et al.Development and evaluation of a sublingual tablet based on recombinant Bet v 1 in birch pollen-allergic patients.Allergy. 2015; 70: 795-804Crossref PubMed Scopus (61) Google Scholar but sublingual treatment will require multiple administrations and therefore remains an inconvenient form of treatment. It is also possible that sublingual application of rBet v 1 will induce oral allergy syndrome as a possible side effect. Finally, SLIT with rBet v 1 can strongly boost allergen-specific IgE production, as was observed for grass pollen allergen extract–containing tablets.30Durham S.R. Yang W.H. Pedersen M.R. Johansen N. Rak S. Sublingual immunotherapy with once-daily grass allergen tablets: a randomized controlled trial in seasonal allergic rhinoconjunctivitis.J Allergy Clin Immunol. 2006; 117: 802-809Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar Currently available data for recombinant birch pollen and recombinant grass pollen allergens thus suggest that recombinant allergens can replace natural allergen extracts.47Pauli G. Larsen T.H. Rak S. Horak F. Pastorello E. Valenta R. et al.Efficacy of recombinant birch pollen vaccine for the treatment of birch-allergic rhinoconjunctivitis.J Allergy Clin Immunol. 2008; 122: 951-960Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 49Jutel M. Jaeger L. Suck R. Meyer H. Fiebig H. Cromwell O. Allergen-specific immunotherapy with recombinant grass pollen allergens.J Allergy Clin Immunol. 2005; 116: 608-613Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 50Klimek L. Schendzielorz P. Pinol R. Pfaar O. Specific subcutaneous immunotherapy with recombinant grass pollen allergens: first randomized dose-ranging safety study.Clin Exp Allergy. 2012; 42: 936-945Crossref PubMed Scopus (47) Google Scholar They offer the advantage of being pharmaceutically well-defined allergens that can be easily produced, but side effects will remain similar as for natural allergen extracts. In addition, it will not be possible to reduce the numbers of treatments, and treatment will remain as inconvenient as for allergen extracts. Furthermore, recombinant wild-type allergens boost allergen-specific IgE production and therefore might not be suitable for preventive vaccination. One possibility to reduce side effects is the genetic engineering of recombinant hypoallergenic allergen derivatives or the chemical denaturation of recombinant allergens. For genetic engineering, many different approaches, such as production of recombinant allergen fragments, mutated allergens, and mosaic approaches, have been used.51Linhart B. Valenta R. Mechanisms underlying allergy vaccination with recombinant hypoallergenic allergen derivatives.Vaccine. 2012; 30: 4328-4335Crossref PubMed Scopus (57) Google Scholar These modifications aim to reduce IgE reactivity and retain T-cell epito