Allergen hybrids - next generation vaccines for Fagales pollen immunotherapy.

BACKGROUND: Trees belonging to the order of Fagales show a distinct geographical distribution. While alder and birch are endemic in the temperate zones of the Northern Hemisphere, hazel, hornbeam and oak prefer a warmer climate. However, specific immunotherapy of Fagales pollen-allergic patients is mainly performed using birch pollen extracts, thus limiting the success of this intervention in birch-free areas. OBJECTIVES: T cells are considered key players in the modification of an allergic immune response during specific immunotherapy (SIT), therefore we thought to combine linear T cell epitope-containing stretches of the five most important Fagales allergens from birch, hazel, alder, oak and hornbeam resulting in a Fagales pollen hybrid (FPH) molecule applicable for SIT. METHODS: A Fagales pollen hybrid was generated by PCR-based recombination of low IgE-binding allergen epitopes. Moreover, a structural-variant FPH4 was calculated by in silico mutagenesis, rendering the protein unable to adopt the Bet v 1-like fold. Both molecules were produced in Escherichia coli, characterized physico-chemically as well as immunologically, and tested in mouse models of allergic sensitization as well as allergy prophylaxis. RESULTS: Using spectroscopic analyses, both proteins were monomeric, and the secondary structure elements of FPH resemble the ones typical for Bet v 1-like proteins, whereas FPH4 showed increased amounts of unordered structure. Both molecules displayed reduced binding capacities of Bet v 1-specific IgE antibodies. However, in a mouse model, the proteins were able to induce high IgG titres cross-reactive with all parental allergens. Moreover, prophylactic treatment with the hybrid proteins prevented pollen extract-induced allergic lung inflammation in vivo. CONCLUSION: The hybrid molecules showed a more efficient uptake and processing by dendritic cells resulting in a modified T cell response. The proteins had a lower IgE-binding capacity compared with the parental allergens, thus the high safety profile and increased efficacy emphasize clinical application for the treatment of Fagales multi-sensitization.

Vaccination against Her-2/neu, with focus on peptide-based vaccines.

Immunotherapy has been a milestone in combatting cancer, by complementing or even replacing classic treatments like surgery, chemotherapy, radiation, and anti-hormonal therapy. In 15%-30% of breast cancers, overexpression of the human epidermal growth factor receptor 2 (Her-2/neu) is associated with more aggressive tumor development. Passive immunization/immunotherapy with the recombinantly produced Her-2/neu-targeting monoclonal antibodies (mAbs) pertuzumab and trastuzumab has been shown to effectively treat breast cancer and lead to a significantly better prognosis. However, allergic and hypersensitivity reactions, cardiotoxicity, development of resistance, lack of immunological memory which results in continuous application over a long period, and cost-intensiveness are among the drawbacks associated with this treatment. Furthermore, intrinsic or acquired resistance is associated with the application of therapeutic mAbs, leading to the disease recurrence. Conversely, these drawbacks could be potentially overcome by vaccination, i.e. an active immunization/immunotherapy approach by activating the patient's own immune system to target cancer, along with inducing immunological memory. This review aims to summarize the main approaches investigated and undertaken for the production of Her-2/neu vaccine candidates, with the main focus on peptide-based vaccines and their evaluation in clinical settings.

Neonatal colonization of mice with Lactobacillus plantarum producing the aeroallergen Bet v 1 biases towards Th1 and T-regulatory responses upon systemic sensitization.

BACKGROUND: The use of recombinant lactic acid bacteria (LAB) as vehicles for mucosal delivery of recombinant allergens is an attractive concept for antigen-defined allergy prevention/treatment. Interventions with LAB are of increasing interest early in life when immune programming is initiated. Here, we investigated the effect of neonatal colonization with a recombinant LAB producing the major birch pollen allergen Bet v 1 in a murine model of type I allergy. METHODS: We constructed a recombinant Lactobacillus (L.) plantarum NCIMB8826 strain constitutively producing Bet v 1 to be used for natural mother-to-offspring mono-colonization of germ-free BALB/c mice. Allergen-specific immunomodulatory effects of the colonization on humoral and cellular immune responses were investigated prior and after sensitization to Bet v 1. RESULTS: Mono-colonization with the Bet v 1 producing L. plantarum induced a Th1-biased immune response at the cellular level, evident in IFN-γ production of splenocytes upon stimulation with Bet v 1. After sensitization with Bet v 1 these mice displayed suppressed IL-4 and IL-5 production in spleen and mesenteric lymph node cell cultures as well as decreased allergen-specific antibody responses (IgG1, IgG2a, and IgE) in sera. This suppression was associated with a significant up-regulation of the regulatory marker Foxp3 at the mRNA level in the spleen cells. CONCLUSION: Intervention at birth with a live recombinant L. plantarum producing a clinically relevant allergen reduces experimental allergy and might therefore become an effective strategy for early intervention against the onset of allergic diseases.

Emerging targets for anticancer vaccination: PD-1.

Among the mechanisms by which tumor cells escape the immune surveillance, one is the interaction between programmed cell death protein 1 (PD-1) and its ligand programmed death-ligand 1 (PD-L1). Inhibition of the PD-1/PD-L1 pathway with monoclonal antibodies as immune checkpoint inhibitors targeting PD-1 or its ligand, PD-L1, represents a milestone in cancer therapy. The application of these antibodies, however, suffers from drawbacks including failure to show a response or benefit in a majority of patients following monotherapy or combination therapy, their frequent administration, and cost intensiveness. Small peptides capable of interfering with PD-1/PD-L1 interaction represent interesting alternatives to antibody-based immune checkpoint inhibitors. Moreover, peptides representing PD-1 or PD-L1 sequences can be used in active immunization approaches to induce antibodies that enhance antitumor immunity by effectively preventing PD-1-mediated inhibition in the host. Importantly, such peptides can readily be combined with peptides derived from cancer antigens to effectively induce an antitumor immune response. In this review, we have summarized the recent developments in the use of small molecules and peptides either to directly block PD-1/PD-L1 interaction, or in vaccination approaches to induce antibody responses stimulating anticancer immunity by blocking PD-1-mediated T-cell inhibition.

Medical students' knowledge and attitudes regarding vaccination against measles, influenza and HPV. An international multicenter study.

INTRODUCTION: Inaccurate information leads to increased scepticism concerning vaccinations among health care workers. Therefore, a proper education of medical students on vaccination is important. METHODS: During summer term 2015, we performed a paper-based survey to identify the knowledge and attitudes of medical students on vaccinations against measles, influenza and HPV in seven medical schools in Germany, Austria and Switzerland. RESULTS: Altogether, 3,652 questionnaires were analyzed. Knowledge of country-specific public recommendations increased significantly with the number of semesters of medical studies. Concerning the knowledge about vaccinations against measles, influenza and HPV, one third of the answers were given correctly. Again, a strong correlation between the knowledge and the semester of medical studies could be observed. The attitudes concerning vaccinations in general and especially for HCWs were highly positive. CONCLUSIONS: This study provides some important arguments for the development of a comprehensive vaccination education for medical students.

E. coli Nissle 1917 is a safe mucosal delivery vector for a birch-grass pollen chimera to prevent allergic poly-sensitization.

Allergic poly-sensitization affects a large number of allergic patients and poses a great challenge for their treatment. In this study we evaluated the effects of the probiotic Escherichia coli Nissle 1917 (EcN) expressing a birch and grass pollen allergen chimera 'Bet v 1, Phl p 1 and Phl p 5' (EcN-Chim) on allergy prevention after oral or intranasal application in poly-sensitized mice. In contrast to oral application, intranasal pretreatment with EcN-Chim prior to poly-sensitization led to a significant reduction of lung inflammation (eosinophils, IL-5, and IL-13 in bronchoalveolar lavage) along with suppressed levels of allergen-specific serum IgE. The suppression was associated with increased levels of allergen-specific IgA in lungs and serum IgG2a along with increased Foxp3, TGF-ß, and IL-10 mRNA in bronchial lymph nodes. In vitro EcN induced high levels of IL-10 and IL-6 in both lung and intestinal epithelial cells. Importantly, using in vivo imaging techniques we demonstrated that intranasally applied EcN do not permanently colonize nose, lung, and gut and this strain might therefore be a safe delivery vector against allergy in humans. In conclusion, our data show that intranasal application of recombinant EcN expressing a multiallergen chimera presents a novel and promising treatment strategy for prevention of allergic poly-sensitization.

Correction: E. coli Nissle 1917 is a safe mucosal delivery vector for a birch-grass pollen chimera to prevent allergic poly-sensitization.

Following the publication of this article, the authors have requested that the Acknowledgements section be amended to include the financing source of the study. The correct Acknowledgments should be as follows: We gratefully acknowledge funding from the Austrian Science Fund SFB F46 and DK MCCA W1248-B30, OeAD-GmbH grants (FR13/2016) and from the Amadeus partnership Hubert Curein French and Austrian program. We thank the BioImaging Center of Lille (Frank Lafont) for the use of the IVIS Lumina XR. We gratefully thank Katharina Ambroz, Elke Korb, Karin Baier, Erika Garner-Spitzer, Joshua Tobias, Gwenaëlle Verbrugghe, and Jéremy Desramaut for their technical assistance and helpful discussions.

Airway inflammation induced after allergic poly-sensitization can be prevented by mucosal but not by systemic administration of poly-peptides.

BACKGROUND: Patients with multiple sensitizations require alternative forms of treatment, as the efficacy of conventional immunotherapy is unsatisfactory. OBJECTIVE: In the present study, we sought to compare the efficacy of a subcutaneously (s.c.) and a mucosally applied polyvalent vaccine to reduce allergic immune responses within airway and lung tissues. METHODS: Female BALB/c mice were intraperitoneally immunized with recombinant (r)Bet v 1, rPhl p 1 and rPhl p 5, followed by an aerosol challenge of birch and phleum pollen extract. For tolerance induction, either a mixture of the immunodominant peptides or a hybrid peptide of the respective antigens was s.c. injected or intranasally applied before poly-sensitization. RESULTS: Mucosal but not systemic pre-treatment with poly-peptides led to significant suppression of eosinophils and IL-5 production in bronchoalveolar lavages, as well as IL-5, IL-4, IL-13 and eotaxin levels in lung cell cultures. Lung histology showed a clear reduction of cellular infiltration and mucus production only in intranasally pre-treated mice. In accordance, also the systemic immune response, characterized by IgE-dependent basophil degranulation and IL-4 levels in vitro, was significantly reduced by mucosal antigen application, but only marginally influenced by subcutaneous pre-treatment. Both treatment routes led to up-regulated CTLA4 expression in splenocytes, whereas only after mucosal pre-treatment Foxp3 expression levels were enhanced in lung CD3(+) T cells. Furthermore, intranasal but not subcutaneous application of the peptides enhanced IL-10 levels in the lungs, indicating regulatory mechanisms operating in local tolerance induction. CONCLUSION: Mucosal application of peptides is superior to systemic application in preventing both local and systemic poly-allergic T helper2 immune responses, suggesting mucosal tolerance induction as an attractive strategy for the primary and secondary prevention of allergic multi-sensitization and lung pathology.

Carbohydrate-based particles reduce allergic inflammation in a mouse model for cat allergy.

BACKGROUND: Allergen-specific immunotherapy (ASIT) is the only treatment of allergic disease that gives long-lasting relief of symptoms. However, concerns for safety and efficiency have highlighted the need for improvement of the therapy. We have previously suggested carbohydrate-based particles (CBPs) as a novel adjuvant and allergen carrier for ASIT. Our aim of this study was to evaluate the therapeutic potential of CBPs in ASIT, employing a mouse model for cat allergy. METHODS: BALB/c mice were subcutaneously immunized with the recombinant (r) cat allergen Fel d 1 followed by intranasal challenge with cat dander extract (CDE). The sensitized mice were therapeutically treated with rFel d 1 covalently coupled to CBPs (CBP-rFel d 1). Airway hyper-reactivity (AHR), infiltration of leucocytes in bronchoalveolar lavage (BAL) fluid, allergen-specific serum immunoglobulin levels and in vitro splenocyte responses were evaluated. RESULTS: Mice treated with CBP-rFel d 1 showed reduced features of allergic inflammation. They responded with (i) significantly decreased AHR and infiltration of eosinophils in BAL fluid after CDE challenge, (ii) the serum level of rFel d 1-specific IgE was reduced and the level of IgG(2)a was more pronounced after CBP-rFel d 1 treatment, and (iii) there was also a tendency of decreased allergen-specific cellular response. CONCLUSIONS: Carbohydrate-based particles are effective tools as adjuvant and allergen carriers for use in ASIT and constitutes a promising strategy to improve allergy treatment.

Monovalent fusion proteins of IgE mimotopes are safe for therapy of type I allergy.

By screening phage display random peptide libraries with purified immunoglobulin E (IgE) from birch pollen-allergic patients, we previously defined peptides mimicking natural IgE epitopes (mimotopes) of the major birch pollen allergen Bet v 1. The present study aimed to define a monovalent carrier for the IgE mimotopes to induce protective antibodies directed to the IgE epitopes, suitable for mimotope-specific therapy. We expressed the selected mimotopes as fusion proteins together with streptococcal albumin binding protein (ABP). The fusion proteins were recognized specifically by anti-Bet v 1 human IgE, which demonstrated that the mimotopes fused to ABP resemble the natural IgE epitope. Bet v 1-specific IgG was induced by immunization of BALB/c mice with fusion proteins. These IgG antibodies could inhibit IgE binding to Bet v 1. Skin testing of Bet v 1 allergic mice showed that the ABP mimotope constructs did not elicit type I skin reactions, although they possess IgE binding structures. Our data suggest that IgE mimotopes are safe for epitope-specific immunotherapy of sensitized individuals, when presented in a monovalent form. Therefore, ABP-fused mimotopes are promising candidates for a new type of immunotherapy based on the precise induction of blocking antibodies.

Edible genetically modified microorganisms and plants for improved health.

The development of new strategies for the delivery of vaccine antigens or immune modulators to the mucosal tissue includes innovative approaches such as the use of genetically modified food microorganisms and plants. Even though the 'proof-of-concept' has recently been established for these two systems, key questions mainly related to efficacy and risk of breaking oral tolerance remain to be critically addressed in the immediate future.

Safety and immunogenicity of concomitant vaccination with the cell-culture based Japanese Encephalitis vaccine IC51 and the hepatitis A vaccine HAVRIX1440 in healthy subjects: A single-blind, randomized, controlled Phase 3 study.

In travellers often several pre-departure immunizations are indicated, thus data are needed about possible interactions between vaccines. This Phase 3 study investigated the immunogenicity and safety of IC51 (JE vaccine) and HAVRIX1440 (hepatitis A vaccine) when administered alone or concomitantly to healthy subjects. The immune response was compared between single and concomitant vaccination in terms of geometric mean titre (GMT) and seroconversion rate (SCR) on Days 28 and 56. Immunogenicity was comparable for the 2 vaccines whether given together or separately which suggests that travellers to such regions could receive the vaccinations concomitantly.

The European LABDEL project and its relevance to the prevention and treatment of allergies.

In March 2001, the European Commission funded a 3-year project (contract no. QLK3-CT-2000-00340) under the fifth Framework Programme to develop and test prototype products based on the oral delivery of vaccine and therapeutic agents using harmless lactic acid bacteria (LAB). The project, best known under its acronym LABDEL (for LAB delivery) also included research on LAB fermentation and technological innovations aimed at enhancing the efficiency of LAB delivery systems (1). One of the key scientific objectives was to investigate the possibility to prevent or treat a type I allergic disease using mucosal administration of LAB expressing the pollen allergen Bet v 1. The aim of this paper was to describe the background of the project with reference to a limited selection of articles and recent reviews as well as the results and major conclusions arising from this part of the project.

A recombinant allergen chimer as novel mucosal vaccine candidate for prevention of multi-sensitivities.

BACKGROUND: As conventional immunotherapy is less efficacious in patients with allergic multi-sensitivities compared with mono-sensitized subjects, new intervention strategies are needed. Therefore, an allergen chimer was genetically engineered for treatment of multi-sensitization with birch and grass pollen on the basis of mucosal tolerance induction. METHODS: The major birch pollen allergen Bet v 1 served as a scaffold for N- and C-terminal linkage of the immunodominant peptides of the grass pollen allergens Phl p 1 and Phl p 5 and this new construct was cloned and expressed in Escherichia coli. After purification, physicochemical and immunological characterization the chimer was used for intranasal tolerance induction prior to poly-sensitization with Bet v 1, Phl p 1 and Phl p 5. RESULTS: The immunological characterization revealed that the conformation of Bet v 1 within the chimer was comparable to that of natural as well as recombinant Bet v 1. The chimer was immunogenic in mice for T and B cell responses to the three allergens. Intranasal application of the chimer prior to poly-sensitization significantly suppressed humoral and cellular allergen-specific Th2 responses and prevented development of airway inflammation upon allergen challenge. Moreover, local allergen-specific IgA antibodies were induced by the chimer. The mechanisms of poly-tolerance induction seemed to be mediated by regulatory cytokines, since TGF-beta and IL-10 mRNA in splenocytes were upregulated and tolerance was transferable with these cells. CONCLUSION: The data indicate that such allergen chimers harboring several unrelated allergens or allergen peptides could serve as mucosal polyvalent vaccines for prevention of multi-sensitivities.

Modulation of allergic immune responses by mucosal application of recombinant lactic acid bacteria producing the major birch pollen allergen Bet v 1.

BACKGROUND: Probiotic lactic acid bacteria (LAB) are able to modulate the host immune system and clinical trials have demonstrated that specific strains have the capacity to reduce allergic symptoms. Therefore, we aimed to evaluate the potential of recombinant LAB producing the major birch pollen allergen Bet v 1 for mucosal vaccination against birch pollen allergy. METHODS: Recombinant Bet v 1-producing Lactobacillus plantarum and Lactococcus lactis strains were constructed. Their immunogenicity was compared with purified Bet v 1 by subcutaneous immunization of mice. Intranasal application of the live recombinant strains was performed to test their immunomodulatory potency in a mouse model of birch pollen allergy. RESULTS: Bet v 1 produced by the LAB was recognized by monoclonal anti-Bet v 1 and IgE antibodies from birch pollen-allergic patients. Systemic immunization with the recombinant strains induced significantly lower IgG1/IgG2a ratios compared with purified Bet v 1. Intranasal pretreatment led to reduced allergen-specific IgE vs enhanced IgG2a levels and reduced interleukin (IL)-5 production of splenocytes in vitro, indicating a shift towards non-allergic T-helper-1 (Th1) responses. Airway inflammation, i.e. eosinophils and IL-5 in lung lavages, was reduced using either Bet v 1-producing or control strains. Allergen-specific secretory IgA responses were enhanced in lungs and intestines after pretreatment with only the Bet v 1-producing strains. CONCLUSIONS: Mucosal vaccination with live recombinant LAB, leading to a shift towards non-allergic immune responses along with enhanced allergen-specific mucosal IgA levels offers a promising approach to prevent systemic and local allergic immune responses.

The role of Foxp3+ T cells in long-term efficacy of prophylactic and therapeutic mucosal tolerance induction in mice.

BACKGROUND: Mucosal tolerance induction is suggested as treatment strategy for allergic diseases. Using a murine model of birch pollen (BP) allergy we investigated the long-term efficacy and the underlying mechanisms of mucosal tolerance induction with two structurally different molecules in a prophylactic and in a therapeutic set-up. METHODS: The three-dimensional major BP allergen Bet v 1 or a nonconformational hypoallergenic fragment thereof was intranasally applied before (prophylaxis) or after sensitization (therapy). RESULTS: In the prophylactic application both the Bet v 1 allergen and the fragment prevented allergic sensitization, and this effect lasted for 1 year. In the therapeutic approach established allergic immune responses were also suppressed after treatment with either of the molecules. However, a long-lasting curative effect (6 months) was only achieved with the Bet v 1 allergen but not with the Bet v 1 fragment. Real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis of splenocytes revealed that tolerance induction with the Bet v 1 allergen was associated with enhanced expression of transforming growth factor (TGF)-beta, interleukin (IL)-10, and Foxp3 mRNA in CD4+ T cells, whereas treatment with the fragment led to the induction of either Foxp3 (prophylaxis) or IL-10 (therapy) alone. CONCLUSION: From these data we concluded (i) that the mechanisms underlying peripheral tolerance are linked to the conformation of the antigen, (ii) that mucosal tolerance is mediated by separate regulatory cell subsets, and (iii) that the long-term efficacy of immunosuppression is associated with the presence of Foxp3+ T cells.