Advantages of recombinant allergens over traditional allergen extracts
Allergic diseases are manifested in susceptible individual by exposure to proteins named allergens that induce an immune response mediated by IgE antibody. Numerous allergens from different sources such as plants, insects, mites and mammals have been obtained as recombinant molecules by molecular cloning. These types of molecules have shown molecular, functional and immunological properties similar to the corresponding natural allergens and, therefore, could be used for in vitro and in vivo diagnosis test of allergy. An important step was done with the development of variants of allergens with reduced allergenicity and preserved immunogenicity, which paved the way toward its rational use in allergen specific immunotherapy to treat allergies. Few of the allergens cloned have been developed to a stage at which they are suitable for use in clinical studies. However, today the academic and scientific communities note a broad and important activity to offer in the near future preparations with enhanced clinical efficacy and safety. In this work, basic aspects and experimental and clinical results of this process are presented.
2. Progress in the molecular cloning and production of allergens
The molecular cloning has provided a practical and efficient way to obtain highly purified molecules for different purposes; in the biomedical sciences this is evident by the increasing amount of biological products, obtained by recombinant DNA technology, which are commercially available for diagnosis and treatment of different diseases, as well as the wide variety of reagents for basic research. The era of molecular cloning of allergen molecules was initiated in 1988 with the report of a cDNA clone coding for the allergen Der p 1 isolated from a cDNA library of the house dust mite
The eukaryotic expression system have the capacity of performing many of the post-translational modifications including signal sequences, disulfide bond formation, and addition of lipid and carbohydrates. A variety of eukaryotic expression systems like yeast, insect cells, mammalian cells and plants are available. The yeast
3. Current vaccines for allergic diseases
Allergies are inflammatory diseases characterized by a Th2 biased response induced in atopic individuals for exposure to allergens. The Th2 response is also induced by helminthes, which occur in an environment characterized by the presence of IL-4, IL-5 and IL-13. Nuocytes [33, 34], basophiles  and type 2 multi-potent progenitor cells  seem to be an important source of this cytokines and necessary for the development of allergic response. Allergen-specific IgE antibodies produced by B cells bind to Fc epsilon receptor 1 (FcεRI) on basophiles or mast cells, sensitizing them. After consecutive exposure, allergen binds to IgE on these cells leading to the release of inflammatory mediators of immediate-type symptoms of allergic diseases and paves the way for late-phase inflammatory responses caused by basophiles, eosinophils and T cells. Allergen specific Th1, Th9, Th17 and Treg cells are also produced in this process [37, 38].
Allergen-specific immunotherapy (SIT) is the only curative and specific approach for treatment of allergies [39, 40]. The current SIT consists of gradual administration of increasing amounts of allergenic extract with the aim to avoid allergic symptoms associated to the exposition. The induction of allergen tolerance is the essential immunological mechanisms of SIT, and involve allergen-specific memory T and B-cell that lead to immune tolerance characterized by a specific noninflammatory reactivity to a given allergen and prevention of new sensitizations and progression of allergic disease. During the immunotherapy, different regulatory and effectors components of the immune system are involved (Figure 1). Allergen tolerance is characterized by the generation of two subgroups of Treg cells: FOXP3+ CD4+ CD25+ Treg cells and inducible Treg cells . T-regulatory type 1 (Tr1) cells have shown to play a major role in allergen tolerance induced by SIT [42, 43]. The immunosuppressor mechanism of Treg cells is mediated by the production of high level of anti-inflammatory cytokines IL-10 and TGF-β, although IFN-γ could also be produced [44-46]. The expression of different subtypes of antibodies during SIT is mediated by the activity of regulatory cytokines secreted by Treg cells; IL-10 is a potent suppressor of allergen-specific IgE and simultaneously increases IgG4 production . SIT increase 10 to 100 folds the serum levels of allergen-specific IgG1 and IgG4 [43, 47]. The IgG4 seems to act as a blocking antibody that interacts with the allergen, avoiding interaction of allergen with the IgE .
Vaccines composed of whole allergenic extract are complex mixtures of known and unknown material, prepared directly from the allergen source, thus containing allergenic and non-allergenic material and being difficult to standardize [49-51]. Some non-allergenic components have been shown to prime a Th2 response , which offset the efficacy of this type of vaccines. SIT with allergenic extract induce a variety of side effects ranging from local to systemic which in some case may be life-threatening . Moreover, in some preparations the important allergens are not well represented or they exhibit poor immunogenicity . Administration of whole allergenic extracts can induce new IgE specificities against allergens present which were not recognized by the patient before treatment . All these facts decrease the efficacy and safety of the current allergen SIT . Therefore, among new approaches to provide a better treatment for allergic diseases is to develop vaccines based on preparations with a well-defined composition, suitable for a good standardization and very low risk of anaphylaxis, here the recombinant allergens or modification of these represent a good option, they show characteristics that could allow to replace advantageously the whole allergenic extracts , (Table 1).
|Contain undefined components, some of which may promote allergic responses|
|Lack or contain low amounts of important allergens|
|Can be contaminated with unwanted materials or allergens from other sources|
|Cannot be tailored to the patient’s sensitization profile|
|May induce new sensitizations|
|Do not suit the international quality standards for vaccines|
|Cannot be compared between different products or batches|
|Do not allow the precise monitoring and investigation of mechanisms underlying treatment|
|Represent molecules with defined physicochemical and immunologic properties that can be modified to foster advantageous characteristics|
|Amounts can be easily controlled on the basis of mass units|
|Potencies and ratios can be exactly adjusted for each molecule|
|Represents pure molecules|
|Vaccines can be exactly tailored according to the patient’s sensitization profile|
|Fit the international quality standards for vaccines|
|Can be precisely compared to give consistent and reproducible products or batches|
|Allow the precise monitoring and investigation of mechanisms underlying treatment|
|Can be reproducible modified to suit different treatment strategies|
4. Recombinant allergens for diagnosis and allergen-specific immunotherapy
Recombinant allergens may be obtained with the same structural and immunological properties of its natural equivalent, therefore, the usefulness for diagnosis or immunotherapy is guarantee. These can be expressed in large amounts in
4.1. Diagnosis of allergy
A more appropriate diagnostic of allergic diseases would be obtained by identification of the particular molecules involved in allergic response, which could be done using purified wild type or recombinant allergens in order to define the sensitization profile of each allergic subject, the concept “Component-resolved diagnosis” was applied to this kind of diagnosis , that would allow a “component-resolved immunotherapy”, in which only the allergens involved in the sensitization are applied to an allergic subject, avoiding new sensitizations. There are some illustrative examples of the goodness of this future practice: in skin tests with three recombinant cherry allergens, rPru av 1, rPru av 3 and rPru av 4, the diagnosis of allergic population could be obtained with sensitivity similar to that obtained with the allergenic extract . The population allergic to peanut was identified using three recombinant peanut allergens (rAra h 1, rAra h 2 and rAra h 3) , in this study and another with celery allergens was demonstrated that recombinant allergens improve the sensitivity of diagnosis compared to allergenic extracts [58, 59]. In allergies with a high compromise of cross-reactivity such as the pollen-related food allergy the power of
The technology of microarrays can be applied to target protein interactions and the serological immune response to antigens . Microarrays are highly useful for detecting all antibodies isotypes and are a powerful tool for component-resolved diagnosis. The primary advantage of microarrays is that specific IgE to thousands allergens can be assayed in parallel with small amounts of serum, at the same time, much less amount of allergen is required. The advantages of protein microarrays to detect specific-antibodies against multiple targets have been taken to develop component-based diagnosis tools. A microarray based test developed by VBC Genomic and Phadia market as “ISAC” that uses a combination of 103 purified natural and recombinant allergens from 47 species, is available in Europe, however, in the United States it has not yet been approved for use by the US Food and Drug Administration and is available only as a research tool (Available at: http://www.pirllab.com/). One of its potentials lies in the recognition of individual patterns of IgE reactivity to protein families with homologues across plant or animal species [66, 67]. When microarray test for diagnosis of birch and timothy allergy were compared with other
To evaluate the clinical significance and allergenicity of several recombinant allergens from
4.2. Allergen-specific immunotherapy
Allergen SIT with recombinant allergens was proposed when it was demonstrated that these molecules have similar or the same biological properties of their natural counterparts [73, 74], and the necessity of highly purified and well standardized allergens were required for overcome the problems related to difficult standardization and management of the doses observed with the whole allergenic extracts. A study with the recombinant pollen allergen Bet v 1 (rBet v 1) demonstrated that immunotherapy with a single allergen is effective for the specific treatment of allergy . In a multicenter, double-blind, placebo-controlled clinical trial, patients with history of birch pollen–related rhinoconjunctivitis were divided in four groups and treated for two years with rBet v 1, natural birch pollen extract, natural Bet v 1 (nBet v 1) or placebo, to compare the efficacy of each preparation for allergen-specific immunotherapy. Treatment with rBet v 1 reduced symptoms of rhinoconjunctivitis and skin reactivity induced by birch pollen, and showed to be safety without serious adverse events. In contrast, one adverse event appears in the group treated with nBet v 1. Clinical improvement and reduction of sensitivity were accompanied with marked increase in Bet v 1-specific IgG1, IgG2 and IgG4 levels, which were higher in the rBet v 1-treated group than in nBet v 1-treated group. Importantly, new IgE specificities were induced in 3 patients treated with birch pollen extract, but in none of rBet v 1 or nBet v 1 treated patients.
In a placebo controlled immunotherapy study, a mixture of equimolar concentration of five
5. Approaches for an immunotherapy of allergy based on modified recombinant allergens
Some allergens are present in the nature as a mix of several isoforms with high structural homology but different IgE reactivity. The production by molecular cloning of natural isoforms with low IgE reactivity has been used to propose anti-allergy vaccines. Bet v 1.0401 and Bet v 1.1001 are isoforms that have lower IgE reactivity compared to the Bet v 1.0101 [77, 78]. Antibody response against Bet v 1.0401 is IgG4-specific and has low capacity to induce basophile degranulation .
The availability of multiple clones of recombinant allergens has facilitated the implementation of site directed mutagenesis to obtain modified allergens for a better immunotherapy. There are several examples of this approach that illustrate the potential use for the development of new vaccines. Mouse allergic individuals are sensitized mainly against the major allergen Mus m 1 a urinary protein belonging to the lipocalin superfamily which have typicall β-barrel fold, that can be modified by mutation in the Tyr 120 residue, [80, 81]. Two hypo-allergenic variants of this allergen; mutants 1Y120A, and Mus m 1-Y120F were expressed in
Hybrid proteins are structures composed by two or more allergens or short portions of them in only one molecule, in this way new interaction and bonds are generated, which may alter the 3D structure and B epitopes characteristic of natural allergens. Decreasing the capacity of IgE binding and mast cell degranulation. However, if these proteins conserve the T cell epitopes, they could induce a protective response after allergen challenge. A single molecule composed by different allergen polypeptides might reduce the number of molecules to be included in the vaccine. Furthermore, hybrid molecules consisting of several copies of homologous allergens or immunologically unrelated allergens could be used for the allergy treatment in the patients who are sensitized to several allergens.
T. P. King, who constructed a molecule composed by two allergens from insect and demonstrated
The utility of hybrid proteins for the immunotherapy of house dust mite allergy have been studied by Asturias,
Recently, a hybrid protein composed of three allergens of
Mosaic proteins are constituted by different segments of the same allergen, in different order as they are present in the native molecule, such re-arrange generate new intra-molecular interactions that alter B cell epitopes. A mosaic protein called P1m constructed with four segments of the pollen allergen Phl p 1, showed lower IgE reactivity compared to the natural allergen and was unable to induce histamine release from basophiles of allergic individuals. However, this molecule conserved capacity to induce the proliferation of PBMCs. Immunized rabbits expressed IgG antibodies that blocked the binding of Phl p 1 to the IgE and inhibit histamine release from basophiles obtained from allergic individuals . Other mosaic protein constructed with segments derived from Phl p 2 reassembled in altered order and expressed as a trimer showed absence of IgE reactivity with sera from allergic patients. Basophile activation and skin prick tests, showed reduction of the allergenicity of this molecule compared to recombinant Phl p 2. Furthermore, IgG antibodies produced by immunized mice were able to inhibit the binding of recombinant Phl p 2 to the IgE from allergic subjects . Mosaic proteins have been studied as a potential vaccine for immunotherapy of birch allergy  and house dust mite allergy . A mosaic protein composed of reorganized segments of Bet v 1 preserved the specific T cell epitopes and showed approximately 100-fold reduced allergenic activity compared with recombinant Bet v 1 [94, 95] and induced specific IgG antibodies inhibitors of IgE reactivity to Bet v1 of sera from patients with pollen allergy . The mosaic protein exhibited none IgE reactivity and lower basophile activation. Furthermore, immunization with Bet v 1 derivatives induced IgG antibodies that recognized Bet v 1 and inhibited IgE binding to Bet v 1 .
Fragments of allergens or modification of these, might be poorly immunogenic because they don´t have enough T cell epitopes capable to stimulate a protective immune response. An increase of immunogenicity can be obtained when proteins are made as oligomers which enhance the number of T cell epitopes in the molecule. It has been observed that immunogenicity of Bet v1 increase when obtained as oligomer [97, 98]. By dot-blot analysis and lymphoproliferative responses in PBMCs from birch pollen allergic patients, trimers of re-organized segments of Bet v1 had lower capacity to bind IgE and enhanced capacity to stimulate lymphoproliferation. The CD203c expression analysis showed reduced allergenicity of these oligomers, and when administrated to mice in an immunization scheme, induced the production of high titer of IgG1 antibody, that inhibited human IgE binding to wild type Bet v 1 .
5.1. Molecules to target specific compartments or receptors
It has been suggested that administration of higher allergen doses enhances the efficacy of immunotherapy . However, administration of high doses increases the risk of anaphylaxis. One approach to overcome this problem is to deliver high doses of allergen to B and T cells directly, thus providing higher effective doses to stimulate a protective response, and avoiding the interaction of allergen with IgE antibodies . An allergen vaccine for cat allergy composed of the major cat allergen Fel d 1 fused to the HIV-derived translocation peptide TAT was designed to mediate cytoplasmic uptake of extracellular proteins [102, 103]. Un modified version of this approach, denominated Modular Antigen Translocation (MAT) technology have been developed [104, 105], which consists of allergen fused to a peptide, to direct them to the cytosol, and a truncated human invariant chain (Ii), to target the protein to MHC class II heterodimers assembled in the endoplasmic reticulum and thus circumventing phagosomal uptake and degradation. The allergens Asp f 1, Der p 1, Bet v 1, PLA2 and Fel d 1 fused to MAT, induced lymphoproliferation of PBMCs stimulated
Dendritic cells (DCs) play an important role in the initiation and maintenance of T cell response to allergens. Its role in the type of T cell response generated can be influenced by the maturation state, while mature DCs induce effector T cell responses characterized by Th1 or Th2 response , immature or semi-mature DCs are tolerogenic and have the ability to induce Tregs . DCs express an array of Fc receptors which have the capacity to enhance allergen uptake through internalization of allergen/antibody receptors complexes. When stimulated with allergen, DCs express FcεRI, and activated a signal-transducing cascade involving immunoreceptor tyrosine-based activation motif (ITAM), which result in increased production of proinflammatory cytokines and chemokines, the induction of robust proliferation of allergen-specific T cells and the development of allergic symptoms . DCs also express the receptor FcγRIIb that contains immunoreceptor tyrosine-based inhibition motif (ITIM) which induces inhibitory signaling events. This receptor can co-aggregate with FcεRI that activating a signaling cascade that culminates in inhibition of FcεRI signaling. Under these assumptions, Zhu, D.
A different molecular design was applied to target allergens to CD64 receptor on antigen presenting cells; a fusion protein (H22-Fel d 1) composed by Fel d 1 linked to the variable region of a monoclonal antibody anti-CD64 was designed to stimulate receptor internalization . Flow cytometry analysis showed that H22-Fel d 1 binds to CD64 and reacted with IgE and IgG with similar affinity compared to native allergen.
6. Insect sting allergy
Insect sting allergy are frequently caused by insect stings of the Apidae family (honeybees and bumblebees), those from the Vespidae family (Vespula, Dolichovespula, Vespa and Polistes genera) and, in some regions, also of the Formicidae family (ants). The sting can induce local or systemic IgE-mediated hypersensitivity reactions that can be fatal . Prevalence of systemic reactions caused by insect stings are reported from 0,3% to 7,5% in the United States and Europe [117, 118]. Up to one fifth of these subjects will eventually experience severe life-threatening reactions. Hymenoptera venoms contain protein allergens, as well as non-allergenic components, including toxins, vasoactive amines, acetylcholine, and kinins. Among the multiple allergens in Hymenoptera venoms, two allergens are importan, the phospholipase A2 from of honey bee (
Several studies have demonstrated that immunotherapy for vespid allergy with venom extracts is clinically effective and improve the quality of life and allergic symptoms. This improvement is correlated to a significant decrease of total IgE levels, and increase in specific IgG and IgG4 levels . However, severe and life-threatening anaphylactic side effects may be induced after the administration of crude allergen extracts .
One of the first attempts to obtain safer methods for immunotherapy of insect allergies was made with allergen-derived peptides, containing T-cell epitopes. Peptides derived from the bee allergen Api m 1, were applied to allergic individuals in different immunotherapy schemes.
The use of recombinant venom allergens for allergen specific immunotherapy has been analyzed in animal models. Intranasal administration of the recombinant allergen from wasp venom, rVes v 5, to mice prior to sensitization with natural allergens lead to a significant reduction of the allergic reaction, reduction of specific IgE and IgG2a levels, increase of mRNA levels of IL-10 and TGF-β. Pretreatment with the whole venom was less effective and caused toxic side reactions, suggesting a favorable use of the recombinant protein . Hybrid proteins composed by allergens from bee venom have shown anti-allergenic properties in
Naturally occurring variants of insect allergens could be also useful for specific immunotherapy. For example, the sting of
Despite the promising results observed with recombinant and modified allergens in
7. Recombinant allergy vaccines in clinical phase trials
Clinical trials with recombinant wild type allergens, and modified allergens have been performed (Table 2). The first studies of allergen SIT with purified molecules were done with peptides containing T cell epitopes either from the cat allergen Fel d 1 or from bee-venom-derived phospholipase, administered without adjuvant [122, 123, 129-135]. Such peptides were characterized by its low or none IgE binding capacity. However, they induced late phase systemic side effects in different grades depending in the dose and route of administration [129, 132, 134, 135]. Therapy with T cell peptides does not seem to influence IgE-mediated allergic reactions, in fact, the majority of studies didn’t find evidence of changes in IgE levels or IgE-mediated allergic inflammation furthermore, no induction of IgG response was noted.
Allergic patients under immunotherapy with hypoallergenic preparations of the major birch pollen allergen Bet v 1 adsorbed to aluminum hydroxide as a pre-seasonal treatment for birch pollen allergy in a clinical trial, expressed high levels of IgG1, IgG2 and IgG4 antibodies directed against Bet v 1. These IgG antibodies blocked allergen-induced basophile degranulation and were associated with the ability of patients to tolerate higher allergen concentrations in nasal provocation tests . Immunotherapy with wild-type recombinant Bet v 1 has also been examined for tablet-based sublingual immunotherapy in a phase II, multicenter, double-blind, placebo-controlled, however, this study is still on course and only have been reported good tolerability of the preparation with no serious adverse events and most side effects observed locally .
In a clinical trial, a group of patients with grass pollen allergy was treated with a combination of the major grass pollen allergens (Phl p 1, Phl p 2, Phl p 5a, Phl p 5b and Phl p 6) or with placebo for subcutaneous immunotherapy . Patients treated with the recombinants improve their symptom medication score and had high IgG antibodies levels against natural grass pollen allergens. Several studies of immunotherapy with these mixed allergens have been performed and registered in the National Institutes of Health Clinical trial database (Table 2). Recently, the immunomodulatory properties of MAT-Fel d 1 was studied in a phase I/IIa clinical study . In a randomized double blind trial, intralymphatic immunotherapy (ILIT) with MAT-Fel d 1 in alum was compared with placebo, consisting in 3 injections of each preparation for two months. MAT-Fel d 1 caused reduced skin reactions compared to equimolar concentration of nFel d 1 by intradermal injection, which proved practically painless and reduced drug-related adverse effects compared to placebo group. The IgG4 serum levels in MAT-Fel d 1 treated group increased by a factor of 5.66, while IgG1 and IgE levels didn´t change. After treatment, PBMCs from allergic individuals secreted higher levels of IL-10 when challenged with rFel d 1. Immunotherapy with MAT-Fel d 1 showed to be successful because patients increased their tolerance to nasal challenge, skin prick and dermal test, with cat dander extract. Improvement of quality of life of patients treated with MAT-Fel d 1 was maintained 300 days after immunotherapy.
|Bet v 1 (Birch pollen allergen)||Bet v 1 trimer||SCIT, DBPC, Phase II||2000|||
|Bet v 1 fragments||SCIT, DBPC, Phase II||2000|||
|Bet v 1 folding variant||SCIT, OC, Phase II||2002||NCT00266526|
|SCIT, DBPC, Phase III||2004||NCT00309062|
|SCIT, DBPC, Phase III||2007||NCT00554983|
|SCIT, Immunological and histological evaluation||2009||NCT00841516|
|Recombinant Bet v 1||SCIT, DBPC, Phase II||2002||NCT00410930|
|SLIT, Phase I||2006||NCT00396149|
|SLIT, Phase I||2007||NCT00889460|
|SLIT, DBPC, Phase II||2008||NCT00901914|
|Birch pollen and apple allergens||Bet v 1 / Mal d 1||SCIT, DBPC, Phase II||2011||NCT01449786|
|Mix: Phl p 1, Phl p 2, Phl p 5a, Phl p 5b and Phl p 6||SCIT, DBPC, Phase II||2002|
|SCIT, DBPC, Phase III||2004||NCT00309036|
|SCIT, DBPC, Phase II||2008||NCT0671268|
|SCIT, DBPC, Phase III||2009||NCT01353755|
|SCIT, Phase II||2011||NCT01445002|
|SCIT, DBPC, Phase IIb||2012 (Initiating)||NCT01538979|
|Peanut allergens||Modified Ara h 1, Ara h 2, Ara h 3||Rectal||2009||NCT00850668|
The National Institutes of Health’s clinical trial database contain information about a study that intends to use the recombinant modified peanut allergens Ara h 1, Ara h 2 and Ara h 3 encapsulated in heat/phenol-killed
8. Some considerations for a recombinant based mite allergy vaccine
The prevalence and severity of allergic diseases such as asthma and rhinitis have increased in recent decades , and house dust mite allergy is one of the most common allergies world-wide which affect more than 50% of allergic patients . Several house dust mites species co-exist in tropical and subtropical regions, however in these places the species
Recent studies with hybrid proteins composed by the most important pollen allergens, have suggested that preparations based on molecules containing the B-epitope spectrum of allergenic extracts could be useful for the diagnosis and allergen-specific immunotherapy [64, 86, 87]. We have engineered several fusion proteins composed by segments of different allergens of
For several years the allergen-specific immunotherapy has been successfully done with natural allergenic extracts. However, they are complex mixtures difficult to standardize that might cause local or systemic reactions, compromising the patient’s life. In the last decades, the molecular cloning applied to the study of allergens has allowed obtaining several recombinant allergens from different sources, and their biological and molecular properties elucidated. Component based diagnosis and immunotherapy is now possible by the availability of several recombinant allergens, which represents the best approach to achieve the most efficacious diagnosis and treatment of allergies, based on the sensitization profile and of each patient. Vaccines for allergic diseases based on recombinant allergens or modification of these, that could modulate the immune response against natural allergens toward a protective response, have been proposed. Hypoallergenic molecules obtained by molecular cloning, in different versions like hybrid molecules, oligomers, mosaic proteins or variants obtained by site-directed mutagenesis have been developed and studied by in vitro test, animal model and clinical trial in humans, indicating potential beneficial use in the near future. Recombinant allergens coupled to carriers for directing the molecule to specific cells or intracellular compartments, preventing unwanted side effects and increasing the specificity of the immune response have been explored.
The promising results showed by