**4. Recombinant allergens for diagnosis and allergen-specific immunotherapy**

Recombinant allergens may be obtained with the same structural and immunological prop‐ erties of its natural equivalent, therefore, the usefulness for diagnosis or immunotherapy is guarantee. These can be expressed in large amounts in *E. coli* or eukaryotic systems at low cost and without contaminants, and manipulating the nucleotide sequence of allergens followed by molecular cloning and protein expression, modified version of allergens that preserve the specific T cell recognition of the natural offending molecule but reduced allergenicity, can be obtained providing a good material for allergen vaccine development.

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 *in vitro* tests (Phadia CAP-FEIA and in-house ELISA), a correlation greater than 0.9, with high sensitivity and specificity was obtained [68]. Latex allergy diagnosis is well known to be confounded by a high rate of false positive results when using conventional testing, and positive specific IgE results does not always mirror the clinical situation. A combination of recombinant latex allergens (Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02) on a microarray, was enough to detect individuals allergic to latex with a sensitivity of 80%, and allows discrimination between genuine allergy and sensitization [69]. Recently, a library of 419 overlapping peptides corresponding to the aminoacid sequence of peanut allergens Ara h 1, Ara h 2 and Ara h 3, printed onto glass slides to asses IgE reactivity, was evaluated as a diagnostic tool that could replace the traditional used double-blind, placebo controlled food challenge, that is time consuming, expensive, stressful for the patient and have the risk for potentially life-threatening anaphylactic reaction [70, 71]. Based on the number or peptides that bind IgE and the intensity of the reaction, was possible to distinguish peanut allergic and peanut tolerant individuals with approximately 90% sensitivity and 95% specificity [71].

From Molecular Cloning to Vaccine Development for Allergic Diseases

http://dx.doi.org/10.5772/52821

297

To evaluate the clinical significance and allergenicity of several recombinant allergens from *B. tropicalis* and *D. pteronyssinus* in asthmatic patients from a tropical environment, IgE level were determined in sera from 90 asthmatic patients and 10 healthy controls. In addition, SPT was performed in a selected group of these patients [72]. Three recombinant allergens Der p 1, Der p 2, and Der p 10 were able to detect 93% of *D. pteronyssinus* allergic subjects. No adverse reactions were observed in the allergic or control subjects who were skin tested. We can conclude that recombinant allergens from *B. tropicalis* and *D. pteronyssinus* are useful for *in*

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 [75]. In a multicenter, double-blind, placebo-controlled clinical trial, patients with history of birch pollen–related rhinoconjunctivitis were divided in four

*vitro* and *in vivo* diagnostic tests of mite allergy diseases.

**4.2. Allergen-specific immunotherapy**

### **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 [56], 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 [57]. The population allergic to peanut was identified using three recombinant peanut allergens (rAra h 1, rAra h 2 and rAra h 3) [58], 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 *in vitro* testing using allergenic extracts is very low [60]. Component resolved diagnosis with recombinant allergens result in excellent sensitivity, when applied to allergy to hazelnut that shows cross-reactivity with pollen allergy. Vespid allergy is characterized by cross-reactivity between hymenoptera species, and it has been established that the true source of sensitization must be defined to ensure the efficacy of venom immunotherapy [61]. Monsalve, *et al.* [62], found that in the Mediterranean regions, a compo‐ nent-resolved diagnosis for wasp allergy could be accurately defined using a mixture of the allergens Ves v 1 and Ves v 5 from Vespula spp, and Pol d 1 and Pol d 5 from *Polistes domini‐ lus*. A combination of these four allergens is enough to differentiate the real causative venom in at least 69% of the population. In allergy with a wide spectrum of sensitization profile as the induced by *Phleum pretense* pollens, the use of recombinants is useful to establish a tailor made immunotherapy approach [63]. A hybrid molecule composed of several segments of a grass pollen allergen showed in skin tests on 32 allergic individuals that with only this molecule all the allergic patients can be identified [64].

The technology of microarrays can be applied to target protein interactions and the serological immune response to antigens [65]. 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 *in vitro* tests (Phadia CAP-FEIA and in-house ELISA), a correlation greater than 0.9, with high sensitivity and specificity was obtained [68]. Latex allergy diagnosis is well known to be confounded by a high rate of false positive results when using conventional testing, and positive specific IgE results does not always mirror the clinical situation. A combination of recombinant latex allergens (Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02) on a microarray, was enough to detect individuals allergic to latex with a sensitivity of 80%, and allows discrimination between genuine allergy and sensitization [69]. Recently, a library of 419 overlapping peptides corresponding to the aminoacid sequence of peanut allergens Ara h 1, Ara h 2 and Ara h 3, printed onto glass slides to asses IgE reactivity, was evaluated as a diagnostic tool that could replace the traditional used double-blind, placebo controlled food challenge, that is time consuming, expensive, stressful for the patient and have the risk for potentially life-threatening anaphylactic reaction [70, 71]. Based on the number or peptides that bind IgE and the intensity of the reaction, was possible to distinguish peanut allergic and peanut tolerant individuals with approximately 90% sensitivity and 95% specificity [71].

To evaluate the clinical significance and allergenicity of several recombinant allergens from *B. tropicalis* and *D. pteronyssinus* in asthmatic patients from a tropical environment, IgE level were determined in sera from 90 asthmatic patients and 10 healthy controls. In addition, SPT was performed in a selected group of these patients [72]. Three recombinant allergens Der p 1, Der p 2, and Der p 10 were able to detect 93% of *D. pteronyssinus* allergic subjects. No adverse reactions were observed in the allergic or control subjects who were skin tested. We can conclude that recombinant allergens from *B. tropicalis* and *D. pteronyssinus* are useful for *in vitro* and *in vivo* diagnostic tests of mite allergy diseases.

### **4.2. Allergen-specific immunotherapy**

**4. Recombinant allergens for diagnosis and allergen-specific**

An Integrated View of the Molecular Recognition and Toxinology - From Analytical Procedures to Biomedical

obtained providing a good material for allergen vaccine development.

Recombinant allergens may be obtained with the same structural and immunological prop‐ erties of its natural equivalent, therefore, the usefulness for diagnosis or immunotherapy is guarantee. These can be expressed in large amounts in *E. coli* or eukaryotic systems at low cost and without contaminants, and manipulating the nucleotide sequence of allergens followed by molecular cloning and protein expression, modified version of allergens that preserve the specific T cell recognition of the natural offending molecule but reduced allergenicity, can be

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 [56], 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 [57]. The population allergic to peanut was identified using three recombinant peanut allergens (rAra h 1, rAra h 2 and rAra h 3) [58], 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 *in vitro* testing using allergenic extracts is very low [60]. Component resolved diagnosis with recombinant allergens result in excellent sensitivity, when applied to allergy to hazelnut that shows cross-reactivity with pollen allergy. Vespid allergy is characterized by cross-reactivity between hymenoptera species, and it has been established that the true source of sensitization must be defined to ensure the efficacy of venom immunotherapy [61]. Monsalve, *et al.* [62], found that in the Mediterranean regions, a compo‐ nent-resolved diagnosis for wasp allergy could be accurately defined using a mixture of the allergens Ves v 1 and Ves v 5 from Vespula spp, and Pol d 1 and Pol d 5 from *Polistes domini‐ lus*. A combination of these four allergens is enough to differentiate the real causative venom in at least 69% of the population. In allergy with a wide spectrum of sensitization profile as the induced by *Phleum pretense* pollens, the use of recombinants is useful to establish a tailor made immunotherapy approach [63]. A hybrid molecule composed of several segments of a grass pollen allergen showed in skin tests on 32 allergic individuals that with only this molecule

The technology of microarrays can be applied to target protein interactions and the serological immune response to antigens [65]. Microarrays are highly useful for detecting all antibodies isotypes and are a powerful tool for component-resolved diagnosis. The primary advantage

**immunotherapy**

Applications

296

**4.1. Diagnosis of allergy**

all the allergic patients can be identified [64].

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 [75]. 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 immu‐ notherapy. 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.

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

From Molecular Cloning to Vaccine Development for Allergic Diseases

http://dx.doi.org/10.5772/52821

299

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 *P. pastoris* with a modified fold [82]. The mutants showed low capacity to react with IgE from allergic individ‐ uals and induced lower basophil degranulation than those induced by Mus m 1. In lympho‐ proliferation assays, using cells from mouse allergic individuals the mutants induced similar lymphoproliferation to that induced by Mus m 1. In other study three variants of an allergen from Artemisia (Art v 1), C22S, C47S and C49S [83], showed low IgE reactivity and mediator release from RBL cells. In addition, the variants C49S and C22S induced significantly higher T cell proliferative response in Artemisia allergic patients compared to the obtained with rArt v 1, suggesting a potential utility for the immunotherapy of population allergic to Artemisia. Using a different approach; mutations in residues involved in IgE binding but maintaining the 3D structure of the natural allergen, Spangforth et al. showed that mutants Gln45-Ser and Pro108-Gly of allergen Bet v 1 displayed lower IgE reactivity and induced synthesis of IgG antibodies that block the IgE reactivity against the natural Bet v1 [84]. Unlike the above mentioned studies. The x-ray crystallography structures of the mutants were similar to the natural allergen, indicating that the reduced IgE reactivity is not mediated by an inadequate

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

T. P. King, who constructed a molecule composed by two allergens from insect and demon‐ strated *in vitro* and mice models studies its anti-allergenic properties [85], pioneered the design of hybrid proteins for allergen immunotherapy. Gonzales-Rioja *et al*. [86] obtained by PCRbased engineering a molecule composed by two allergens from the pollen *Parietaria judaica*

treatment in the patients who are sensitized to several allergens.

basophile degranulation [79].

folding.

**Hybrid proteins**

**Hypo-allergens obtained by site-directed mutagenesis**

In a placebo controlled immunotherapy study, a mixture of equimolar concentration of five *Phleum pretense* allergens (Phl p 1, Phl p 2, Phl p 5a, Phl p 5b and Phl p 6) was administered via subcutaneous for 18 months in patients with grass pollen–induced allergic rhinitis, to deter‐ mining efficacy and safety [43]. The immunotherapy showed a 36.5% lower median average symptom score for active treatment compared with placebo and reduction in the need for medication. By the first and second pollen season, improvement in quality of life scores was present in the patients receiving active treatment. Active treatment induced IgG1 concentra‐ tions approximately 60-fold, peaking during the rusty 12 months of the study and IgG4 levels showed a 4000 fold increase by the end of treatment. In contrast, after immunotherapy IgE levels in the active treated group were significantly lower than placebo group. Only about 1% of recombinant grass allergen injections led to systemic reactions. This was the first clinical study of immunotherapy with a cocktail of 5 recombinant grass pollen allergens that showed its clinical efficacy, good tolerance and strong induction of allergen specific IgG antibody response.
