Raffaela Campana

Allergen‐specific immunotherapy: from therapeutic vaccines to prophylactic approaches

Abstract.  Valenta R, Campana R, Marth K, van Hage M (Division of Immunopathology, Department of Pathophysiology and Allergy Research, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna; Christian Doppler Laboratory for Allergy Research, Medical University of Vienna, Vienna, Austria; and Department of Medicine, Clinical Immunology and Allergy Unit, Karolinska Institutet and University Hospital, Stockholm, Sweden) Allergen‐specific immunotherapy: from therapeutic vaccines to prophylactic approaches (Review). J Intern Med 2012; 272: 144–157.

A Combination Vaccine for Allergy and Rhinovirus Infections Based on Rhinovirus-Derived Surface Protein VP1 and a Nonallergenic Peptide of the Major Timothy Grass Pollen Allergen Phl p 1

Allergens and rhinovirus infections are among the most common elicitors of respiratory diseases. We report the construction of a recombinant combination vaccine for allergy and rhinovirus infections based on rhinovirus-derived VP1, the surface protein which is critically involved in infection of respiratory cells, and a nonallergenic peptide of the major grass pollen allergen Phl p 1. Recombinant hybrid molecules consisting of VP1 and a Phl p 1-derived peptide of 31 aa were expressed in Escherichia coli. The hybrid molecules did not react with IgE Abs from grass pollen allergic patients and lacked allergenic activity when exposed to basophils from allergic patients. Upon immunization of mice and rabbits, the hybrids did not sensitize against Phl p 1 but induced protective IgG Abs that cross-reacted with group 1 allergens from different grass species and blocked allergic patients’ IgE reactivity to Phl p 1 as well as Phl p 1-induced basophil degranulation. Moreover, hybrid-induced IgG Abs inhibited rhinovirus infection of cultured human epithelial cells. The principle of fusing nonallergenic allergen-derived peptides onto viral carrier proteins may be used for the engineering of safe allergy vaccines which also protect against viral infections.

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.

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.

Cigarette smoke facilitates allergen penetration across respiratory epithelium

Background:  The association between cigarette smoke exposure and allergic airway disease is a matter for debate. We sought to investigate in an in vitro system whether active smoking reduces the integrity and barrier function of the respiratory epithelium and thus facilitates allergen penetration.

Hypoallergenic derivatives of the major birch pollen allergen Bet v 1 obtained by rational sequence reassembly

BACKGROUND At least 100 million patients suffer from birch pollen allergy. OBJECTIVE Rational design of recombinant derivatives of the major birch pollen allergen, Bet v 1, characterized by reduced IgE reactivity, preservation of sequences relevant for the induction of allergen-specific blocking IgG, and maintenance of T-cell epitopes for immunotherapy of birch pollen allergy. METHODS Three recombinant mosaic proteins derived from Bet v 1 were generated by reassembly of codon-optimized genes coding for Bet v 1 fragments containing the elements for the induction of allergen-specific blocking IgG antibodies and the major T-cell epitopes. The proteins were expressed in Escherichia coli as recombinant mosaic molecules and compared with the Bet v 1 wild-type protein by chemical and structural methods, regarding IgE-binding and IgG-binding capacity, in basophil activation assays and tested for the in vivo induction of IgG responses. RESULTS Three recombinant Bet v 1 (rBet v 1) mosaic proteins with strongly reduced IgE reactivity and allergenic activity were expressed and purified. Immunization with the recombinant hypoallergens induced IgG antibodies that inhibited IgE reactivity of patients with allergy to Bet v 1 comparable to those induced with the rBet v 1 wild-type allergen. CONCLUSION We report the generation and preclinical characterization of 3 hypoallergenic rBet v 1 derivatives with suitable properties for immunotherapy of birch pollen allergy.

Dissection of the IgE and T-cell recognition of the major group 5 grass pollen allergen Phl p 5

BACKGROUND The major timothy grass pollen allergen Phl p 5 belongs to the most potent allergens involved in hay fever and asthma. OBJECTIVE This study characterized immune-dominant IgE- and T-cell-recognition sites of Phl p 5. METHODS Seven peptides, P1 to P7 with a length of 31 to 38 amino acids that spanned the Phl p 5 sequence, were synthesized, characterized by circular dichroism spectroscopy, and tested for IgE reactivity, basophil activation, and T-cell reactivity. Carrier-bound peptides were studied for their ability to induce IgG antibodies in rabbits which recognize Phl p 5 or cross-reactive allergens from different grass species. Peptide-specific antibodies were tested for the capability to inhibit IgE reactivity to Phl p 5 and allergen-induced basophil activation of patients with allergy. RESULTS The peptides exhibited no secondary structure and showed no IgE reactivity or relevant allergenic activity, indicating that Phl p 5 IgE epitopes are conformational. Except for P3, peptide-specific IgG antibodies blocked IgE binding to Phl p 5 of patients with allergy and cross-reacted with temperate grasses. IgE inhibition experiments and molecular modeling identified several clustered conformational IgE epitopes on the N- as well as C-terminal domain of Phl p 5. P4, which stimulated the strongest T-cell and cytokine responses in patients, was not part of the major IgE-reactive regions. CONCLUSION Our study shows an interesting dissociation of the major IgE- and T-cell-reactive domains in Phl p 5 which provides a basis for the development of novel forms of immunotherapy that selectively target IgE or T-cell responses.

Altered IgE epitope presentation: A model for hypoallergenic activity revealed for Bet v 1 trimer.

In order to reduce side effects in the course of allergen specific immunotherapy hypoallergenic allergen derivatives with reduced IgE reactivity have been made by genetic engineering. In contrast to other recombinant hypoallergenic allergen derivatives which showed reduced IgE reactivity, a recombinant trimer of the major birch pollen allergen Bet v 1 showed reduced allergenic activity despite preserved IgE reactivity. We studied rBet v 1 trimer by SDS-PAGE, mass spectrometry, circular dichroism and gel filtration. Furthermore we investigated IgE and IgG reactivity of the rBet v 1 trimer in solid and liquid phase assays and compared its allergenic activity with that of rBet v 1 wildtype using basophil activation assays. In solid phase immunoassays rBet v 1 trimer exhibited even stronger IgE reactivity than the rBet v 1 wildtype, whereas both proteins were equally well recognized by Bet v 1-specific IgG antibody probes. In fluid phase IgE experiments rBet v 1 trimer inhibited IgE reactivity to rBet v 1 wildtype but showed a more than 10-fold reduced allergenic activity compared to the rBet v 1 monomer. By analytical gel filtration it was demonstrated that, despite its monomeric appearance in SDS-PAGE the trimer occurred in fluid phase in the form of defined high molecular weight (>600 kDa) aggregates whereas rBet v 1 wildtype strictly appeared as monomeric protein. The results indicate that the hypoallergenic nature of the rBet v 1 trimer is due to formation of defined high molecular weight aggregates which may be responsible for an altered presentation of IgE epitopes in a form with reduced capacity to crosslink effector-cell bound IgE. We thus provide evidence for a novel mechanism for hypoallergenic activity.

Frequent occurrence of T cell-mediated late reactions revealed by atopy patch testing with hypoallergenic rBet v 1 fragments.

Background Late allergic reactions are common in the course of allergen-specific immunotherapy and even occur with allergy vaccines with reduced IgE reactivity. Objective We sought to study atopy patch test (APT) reactions and T-cell responses to the recombinant birch pollen allergen Bet v 1 and recombinant hypoallergenic T-cell epitope–containing Bet v 1 fragments in patients with birch pollen allergy with and without atopic dermatitis (AD). Methods A clinical study was conducted in 15 patients with birch pollen allergy with AD (group 1), 5 patients with birch pollen allergy without AD (group 2), 5 allergic patients without birch pollen allergy (group 3), and 5 nonallergic subjects (group 4) by performing skin prick tests and APTs with rBet v 1 and hypoallergenic rBet v 1 fragments. T-cell, cutaneous lymphocyte antigen (CLA)+ and CCR4+ T-cell and cytokine responses were studied by thymidine uptake, carboxyfluorescein diacetate succinimidyl ester staining, and Luminex technology, respectively. Results rBet v 1 and hypoallergenic rBet v 1 fragments induced APT reactions in not only most of the patients with birch pollen allergy with AD (11/15) but also in most of those without AD (4/5). Patients with birch pollen allergy with AD had higher Bet v 1–specific proliferation of CLA+ and CCR4+ T cells compared with patients with birch pollen allergy without AD. There were no differences in Bet v 1–specific CLA+ and CCR4+ proliferation and cytokine secretion in patients with and without APT reactions. Conclusion Hypoallergenic rBet v 1 fragments induce T cell–dependent late reactions not only in patients with birch pollen allergy with AD but also in those without AD, which can be determined based on APT results but not based on in vitro parameters.

Recombinant allergy vaccines based on allergen-derived B cell epitopes

Immunoglobulin E (IgE)-associated allergy is the most common immunologically-mediated hypersensitivity disease. It affects more than 25% of the population. In IgE-sensitized subjects, allergen encounter can causes a variety of symptoms ranging from hayfever (allergic rhinoconjunctivitis) to asthma, skin inflammation, food allergy and severe life-threatening anaphylactic shock. Allergen-specific immunotherapy (AIT) is based on vaccination with the disease-causing allergens. AIT is an extremely effective, causative and disease-modifying treatment. However, administration of natural allergens can cause severe side effects and the quality of natural allergen extracts limits its application. Research in the field of molecular allergen characterization has allowed deciphering the molecular structures of the disease-causing allergens and it has become possible to engineer novel molecular allergy vaccines which precisely target the mechanisms of the allergic immune response and even appear suitable for prophylactic allergy vaccination. Here we discuss recombinant allergy vaccines which are based on allergen-derived B cell epitopes regarding their molecular and immunological properties and review the results obtained in clinical studies with this new type of allergy vaccines.

Recombinant allergen and peptide-based approaches for allergy prevention by oral tolerance

Several studies conducted in animal models for immunologically-mediated hypersensitivity diseases have shown that oral administration of antigens early in life can prevent the development of specific humoral and cellular immune responses and thus hypersensitivity reactions to the respective antigens. Such data were also obtained in models for Immunoglobulin E (IgE)-associated allergy, the most common hypersensitivity disease affecting more than 25% of the population. Based on data obtained in animal models for allergy several clinical intervention studies have been conducted in children to study if oral administration of materials containing allergens or allergen-derived peptides early in life can prevent the subsequent development of allergy. In this article we argue that oral tolerance induction could be a potent way to prevent allergy and may be even improved regarding efficacy provided that well-defined allergen molecules and/or allergen-derivatives were used in optimized dose regimens and periods of intervention. The knowledge regarding the molecular and immunological characteristics of allergens which has been achieved in the last decades is a prerequisite for such a treatment. In fact, defined recombinant allergens/allergen derivatives and allergen-derived synthetic peptides from the most common allergen sources are now available for targeted intervention. Moreover, molecular allergy diagnosis allows deciphering the disease-causing relevant allergens for different regions in the world allowing composing cocktails of tolerogens according to the needs of populations from different parts of the world. Furthermore, it is suggested to use defined allergen molecules and epitopes in the analysis of clinical tolerance studies. This will allow understanding if clinical unresponsiveness is due to true immunological tolerance or to other mechanisms such as induction of blocking antibodies or cellular immunomodulation. Using molecularly defined tolerogens it can now be explored if oral tolerance induction is a powerful strategy to prevent IgE-associated allergy.