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The skin has been intensely investigated as a target tissue for immunization because it is populated by multiple types of antigen presenting cells. Directly addressing dendritic cells or Langerhans cells in vivo represents an attractive strategy for inducing T cell responses in cancer immunotherapy. We and others have studied fractional laser ablation as a novel method combining efficient delivery of macromolecules to the skin with an inherent adjuvant effect of laser illumination. In this proof of concept study, we demonstrate the feasibility of peptide delivery to the skin using the P.L.E.A.S.E. professional Erb:YAG fractional infrared laser together with EPIMMUN patches. In an ovalbumin mouse model we demonstrate that a dry patch formulation of SIINFEKL peptide in combination with CpG-ODN1826, but not imiquimod or polyI:C, induces potent cytotoxic T cell responses, which can be further boosted by co-delivery of the pan-helper T cell epitope PADRE.

Patients with spinal cord injury (SCI) frequently develop infections that may affect quality of life, be life-threatening, and impair their neurological recovery in the acute and subacute injury phases. Therefore, identifying patients with SCI at risk for developing infections in this stage is of utmost importance. We determined the systemic levels of immune cell populations, cytokines, chemokines, and growth factors in 81 patients with traumatic SCI at 4 weeks after injury and compared them with those of 26 age-matched healthy control subjects. Patients who developed infections between 4 and 16 weeks after injury exhibited higher numbers of neutrophils and eosinophils, as well as lower numbers of lymphocytes and eotaxin-1 (CCL11) levels. Accordingly, lasso logistic regression showed that incomplete lesions (American Spinal Injury Association Impairment Scale [AIS] C and D grades), the levels of eotaxin-1, and the number of lymphocytes, basophils, and monocytes are predictive of lower odds for infections. On the other hand, the number of neutrophils and eosinophils as well as, in a lesser extent, the levels of IP-10 (CXCL10), MCP-1 (CCL2), BDNF [brain-derived neurotrophic factor], and vascular endothelial growth factor [VEGF]-A, are predictors of increased susceptibility for developing infections. Overall, our results point to systemic immune disbalance after SCI as predictors of infection in a period when infections may greatly interfere with neurological and functional recovery and suggest new pathways and players to further explore novel therapeutic strategies.

The progressive accumulation of misfolded α-synuclein (α-syn) in the brain is widely considered to be causal for the debilitating clinical manifestations of synucleinopathies including, most notably, Parkinson’s disease (PD). Immunotherapies, both active and passive, against α-syn have been developed and are promising novel treatment strategies for such disorders. To increase the potency and specificity of PD vaccination, we created the ‘Win the Skin Immune System Trick’ (WISIT) vaccine platform designed to target skin-resident dendritic cells, inducing superior B and T cell responses. Of the six tested WISIT candidates, all elicited higher immune responses compared to conventional, aluminum adjuvanted peptide-carrier conjugate PD vaccines, in BALB/c mice. WISIT-induced antibodies displayed higher selectivity for α-syn aggregates than those induced by conventional vaccines. Additionally, antibodies induced by two selected candidates were shown to inhibit α-syn aggregation in a dose-dependent manner in vitro. To determine if α-syn fibril formation could also be inhibited in vivo, WISIT candidate type 1 (CW-type 1) was tested in an established synucleinopathy seeding model and demonstrated reduced propagation of synucleinopathy in vivo. Our studies provide proof-of-concept for the efficacy of the WISIT vaccine technology platform and support further preclinical and clinical development of this vaccine candidate.

The skin represents an attractive target tissue for vaccination against respiratory viruses such as SARS-CoV-2. Laser-facilitated epicutaneous immunization (EPI) has been established as a novel technology to overcome the skin barrier, which combines efficient delivery via micropores with an inherent adjuvant effect due to the release of danger-associated molecular patterns. Here we delivered the S1 subunit of the Spike protein of SARS-CoV-2 to the skin of BALB/c mice via laser-generated micropores with or without CpG-ODN1826 or the B subunit of heat-labile enterotoxin of E.coli (LT-B). EPI induced serum IgG titers of 1:3200 that could be boosted 5 to 10-fold by co-administration of LT-B and CpG, respectively. Sera were able to inhibit binding of the spike protein to its receptor ACE2. Our data indicate that delivery of recombinant spike protein via the skin may represent an alternative route for vaccines against Covid-19.

Multifunctional gelatin nanoparticles modified by NIR-emitting gold/silver alloy nanoclusters and loaded with ovalbumin (OVA) as a model antigen were developed. Two different designs of nanoparticles were introduced; positively charged NPs with OVA displayed over the surface (S-NPs) versus OVA encapsulated in the NPs’ matrix and the surface is functionalized by dextran (Dex-NPs) for dendritic cell targeting. The nanoparticles showed average particle sizes of 210 and 305 nm and zeta potentials of +25.6 and −23.9 mV, for S-NPs and Dex-NPs, respectively. Both types of NPs succeeded to induce maturation of murine bone marrow-derived dendritic cells (BMDCs) as indicated by the upregulated surface expression of MHC-II and co-stimulatory molecules CD86, CD80 and CD40. Dex-NPs induced no cytotoxicity in BMDCs, in contrast to S-NPs. Functionalization of NPs with dextran increased their uptake by BMDCs, enhanced secretion of immune stimulatory chemokines, and boosted their T cell stimulation capacity. Co-culture of NP loaded BMDCs with OVA-specific CD4 or CD8 T cells, induced enhanced T cell proliferation and release of IL-2 from both CD8 and CD4 cells and IFN-γ from CD8 T cells. This highlights the potential of the developed NPs as vaccines for inducing T helper 1 type CD4 as well as CD8 responses, such as vaccines for cancer or viral infections.

AbstactIntroductionProtein fold stability has been proposed to represent an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Using in silico prediction, we introduced point mutations in proteins that either increase or decrease their fold-stability without altering immunodominant epitopes or changing the overall structure of the protein. Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization.MethodsUsing the MAESTRO software tool, stabilizing or destabilizing mutations were selected and verified by molecular dynamics simulations. The mutants were expressed in E. coli, purified tag-free, and analyzed for thermal stability and resistance to endolysosomal proteases. The resulting peptides were analysed by degradome assay and mass spectrometry. The structure of the most stable mutant protein was obtained by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model.ResultsFour stabilizing and two destabilizing mutations were identified by MAESTRO. Experimentally determined changes in thermal stability compared to the wild type protein ranged from -5 to +14 °C. Destabilization led to faster proteolytic processing in vitro, whereas highly stabilized mutants were degraded very slowly. However, the overall pattern of identified peptides remained very similar. This was confirmed in bone marrow derived dendritic cells that processed and presented the immune dominant epitope from a destabilized mutant more efficiently. In vivo, stabilization resulted in a shift in immune polarization as indicated by higher levels of IgG2a and increased secretion of TH1/TH17 cytokines.ConclusionMAESTRO was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with susceptibility to protease resistance and presentation of pMHCII on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

BackgroundAllergen-specific immunotherapy via the skin targets an area rich in antigen presenting cells, but can be associated with local and systemic side effect. Allergen-polysaccharide neoglycogonjugates can increase immunization efficacy by targeting and activating dendritic cells via C-type lectin receptors and reduce side effects.ObjectiveWe investigated the immunogenicity, allergenicity and therapeutic efficacy of laminarin-ovalbumin neoglycoconjugates (LamOVA).MethodsThe biological activity of LamOVA was characterizedin vitrousing bone marrow derived dendritic cells. Immunogenicity and therapeutic efficacy was analyzed in BALB/c mice. Epicutaneous immunotherapy (EPIT) was performed using fractional infrared laser ablation to generate micropores in the skin and the effects of LamOVA on blocking IgG, IgE, cellular composition of BAL, lung, and spleen, lung function, and T cell polarization was assessed.ResultsConjugation of laminarin to ovalbumin reduced its IgE binding capacity 5-fold and increased its immunogenitiy 3-fold in terms of IgG generation. EPIT with LamOVA induced significantly higher IgG levels than OVA, matching the levels induced by s.c. injection of OVA/alum (SCIT). EPIT was equally effective as SCIT in terms of blocking IgG induction and suppression of lung inflammation and airway hyperresponsiveness, but SCIT was associated with a higher level of therapy induced IgE and TH2 cytokines. EPIT with LamOVA induced significantly lower local skin reactions during therapy compared to unconjugated OVA.ConclusionConjugation of the allergen to laminarin increased its immunogenicity while at the same time reducing local side effects. LamOVA EPIT via laser generated micropores is safe and equally effective to SCIT with alum, without the need for adjuvant.

BackgroundThe skin resembles an attractive target for vaccination due to its accessibility and abundance of resident immune cells. Cells like γδ T cells and mast cells (MCs) are part of the first line of defence against exogenous threats. Despite being important mediators for eliciting TH2 immune responses after epithelial stress, γδ T cell and MC function still remains to be completely understood. Here, we aimed to characterize their roles in shaping adaptive immune responses after laser-mediated epicutaneous immunization (EPI).Methodsγδ T cell knock out, MC depleted, and wildtype control mice were immunized with mannan-conjugated grass pollen allergen Phl p 5 (P5-MN) by laser-mediated EPI. After 2-3 immunizations, cytokine expression, T helper polarization, and antigen-specific IgG1/IgE levels were analysed. The local cytokine/chemokine milieu after laser microporation was determined.ResultsWhile the majority of inflammatory chemokines and cytokines induced by laser treatment was not affected by the presence of γδ T cells or MCs, RANTES, was elevated in γδ T cell knock out mice, and GROα and TSLP, were significantly decreased after MC depletion. However, absence of γδ T cells or depletion of MC had no substantial effect on adaptive humoral or cellular immune responses after laser-mediated EPI, except for slightly reduced IgG1 and effector T cell levels in MC depleted mice.Conclusionsγδ T cells did not play a pivotal role in shaping the humoral and cellular adaptive immune response after laser-mediated EPI, whereas MC depletion decreased numbers of effector T cells, indicating a potential role of MCs in the activation and maturation of T cells after EPI.HighlightsLaser microporation induces an inflammatory chemokine milieu at the site of immunizationγδ T cells and mast cells contribute to the steady-state or damage-induced cytokine milieu in the skinγδ T cells and mast cells are dispensable for adaptive immunity after laser-mediated immunization

BACKGROUND Skin-based immunotherapy of type 1 allergies has recently been re-investigated as an alternative for subcutaneous injections. In the current study, we employed a mouse model of house dust mite (HDM)-induced lung inflammation to explore the potential of laser-facilitated epicutaneous allergen-specific treatment. METHODS Mice were sensitized against native Dermatophagoides pteronyssinus extract and repeatedly treated by application of depigmented D pteronyssinus extract via laser-generated skin micropores or by subcutaneous injection with or without alum. Following aerosol challenges, lung function was determined by whole-body plethysmography and bronchoalveolar lavage fluid was analyzed for cellular composition and cytokine levels. HDM-specific IgG subclass antibodies were determined by ELISA. Serum as well as cell-bound IgE was measured by ELISA, rat basophil leukemia cell assay, and ex vivo using a basophil activation test, respectively. Cultured lymphocytes were analyzed for cytokine secretion profiles and cellular polarization by flow cytometry. RESULTS Immunization of mice by subcutaneous injection or epicutaneous laser microporation induced comparable IgG antibody levels, but the latter preferentially induced regulatory T cells and in general downregulated T cell cytokine production. This effect was found to be a result of the laser treatment itself, independent from extract application. Epicutaneous treatment of sensitized animals led to induction of blocking IgG, and improvement of lung function, superior compared to the effects of subcutaneous therapy. During the whole therapy schedule, no local or systemic side effects occurred. CONCLUSION Allergen-specific immunotherapy with depigmented HDM extract via laser-generated skin micropores offers a safe and effective treatment option for HDM-induced allergy and lung inflammation.

Allergen-specific immunotherapy is the only curative approach for the treatment of allergies. There is an urgent need for improved therapies, which increase both, efficacy and patient compliance. Novel routes of immunization and the use of more advanced vaccine platforms have gained heightened interest in this field. Areas covered: The current status of allergen-specific immunotherapy is summarized and novel routes of immunization and their challenges in the clinics are critically discussed. The use of nanoparticles as novel delivery system for allergy vaccines is comprehensively reviewed. Specifically, the advantages of silica nanoparticles as vaccine carriers and adjuvants are summarized. Expert opinion: Future allergen-specific immunotherapy will combine engineered hypoallergenic vaccines with novel routes of administration, such as the skin. Due to their biodegradability, and the easiness to introduce surface modifications, silica nanoparticles are promising candidates for tailor-made vaccines. By covalently linking allergens and polysaccharides to silica nanoparticles, a versatile vaccination platform can be designed to specifically target antigen-presenting cells, render the formulation hypoallergenic, and introduce immunomodulatory functions. Combining potent skin vaccination methods, such as fractional laser ablation, with nanoparticle-based vaccines addresses all the requirements for safe and efficient therapy of allergic diseases.

Introduction: In modern vaccinology and immunotherapy, recombinant proteins more and more replace whole organisms to induce protective or curative immune responses. Structural stability of proteins is of crucial importance for efficient presentation of antigenic peptides on MHC, which plays a decisive role for triggering strong immune reactions. Areas covered: In this review, we discuss structural stability as a key factor for modulating the potency of recombinant vaccines and its importance for antigen proteolysis, presentation, and stimulation of B and T cells. Moreover, the impact of fold stability on downstream events determining the differentiation of T cells into effector cells is reviewed. We summarize studies investigating the impact of protein fold stability on the outcome of the immune response and provide an overview on computational methods to estimate the effects of point mutations on protein stability. Expert commentary: Based on this information, the rational design of up-to-date vaccines is discussed. A model for predicting immunogenicity of proteins based on their conformational stability at different pH values is proposed.

Coupling of long chain carbohydrates to allergens can mask IgE epitopes and thus reduce the allergenicity of the neoglycoconjugates. The resulting molecules can target and activate dendritic cells via C-type lectin receptors. Neoglycoconjugates therefore have a high potential for epicutaneous immunotherapy (EPIT) by delivering a hypoallergenic vaccine to a tissue rich in dendritic cells. In the current study we test the therapeutic efficiency of the beta-glucan laminarin conjugated to ovalbumin (LamOVA) in a mouse model of allergic asthma. Sensitized Balb/C mice were treated epicutaneously via micropores created by fractional laser ablation. Humoral and cellular responses were evaluated by ELISA, RBL assay and BAT. Lung function was analyzed by whole body plethysmography. Laminarin remained biologically active after coupling to ovalbumin as indicated by binding to Dectin-1 receptor. IgE epitopes were successfully masked according to in vitro IgE binding aasay and skin reaction scores in therapeutic setup.LamOVA was more potent in stimulating humoral immune responses in comparison to OVA and induced the highest IgG1 and IgG2a production. Epicutaneous treatment with OVA and LamOVA reduced lung inflammation and improved lung function of sensitized mice more efficiently compared to s.c. injection with OVA/alum. Epicutaneous immunotherapy can alleviate lung inflammation and reduce airway hyperresponsiveness in sensitized mice more efficiently than s.c. injection with alum. Coupling of allergen to laminarin reduces local side effects and increases the immunogenicity, resulting in a safer therapy.

Due to the worldwide increase in allergies and a limited efficacy of therapeutic interventions, the need for prophylactic vaccination against allergies has been recognized. mRNA and DNA vaccines have demonstrated their high potential for preventing allergic sensitization by inducing an immunological bias that prevents TH2 sensitization. However, only mRNA vaccines fulfill the stringent safety requirements for vaccination of healthy children. In this chapter, we describe the generation of conventional as well as self-replicating mRNA vaccines and methods to test their prophylactic efficacy in animal models.

Due to the worldwide increase in allergies and a limited efficacy of therapeutic interventions, the need for prophylactic vaccination against allergies has been recognized. mRNA and DNA vaccines have demonstrated their high potential for preventing allergic sensitization by inducing an immunological bias that prevents TH2 sensitization. However, only mRNA vaccines fulfill the stringent safety requirements for vaccination of healthy children. In this chapter, we describe the generation of conventional as well as self-replicating mRNA vaccines and methods to test their prophylactic efficacy in animal models.

Background Methods to deliver an antigen into the skin in a painless, defined, and reproducible manner are essential for transcutaneous immunization (TCI). Here, we employed an ablative fractional infrared laser (P.L.E.A.S.E. Professional) to introduce clinically relevant vaccines into the skin. To elicit the highest possible antibody titers with this system, we optimized different laser parameters, such as fluence and pore number per area, and tested various adjuvants. Methods BALB/c mice were immunized with Hepatitis B surface antigen (HBsAg) by laser-microporation. Adjuvants used were alum, CRM 197 , monophosphoryl lipid A, heat-labile enterotoxin subunit B of E. coli (LT-B), and CpG ODN1826. The influence of different fluences (2.1 to 16.8 J/cm 2 ) and pore densities (5–15%) was investigated. Furthermore, immunogenicity of HBsAg and the commercially available conjugate vaccines ActHIB® and Menveo® applied via TCI was compared to standard i.m. injection. Antigen-specific antibody titers were assessed by luminometric ELISA. Results Antibody titers against HBsAg were dependent on pore depth and peaked at a fluence of 8.4 J/cm 2 . Immunogenicity was independent of pore density. Adjuvantation with alum significantly reduced antibody titers after TCI, whereas other adjuvants only induced marginal changes in total IgG titers. LT-B and CpG shifted the polarization of the immune response as indicated by decreased IgG1/IgG2a ratios. HBsAg/LT-B applied via TCI induced similar antibody titers compared to i.m. injection of HBsAg/alum. In contrast to i.m. injection, we observed a dose response from 5 to 20 μg after TCI. Both, ActHIB® and Menveo® induced high antibody titers after TCI, which were comparable to i.m. injection. Conclusions Alum, the most commonly used adjuvant, is contraindicated for transcutaneous vaccination via laser-generated micropores. TCI with optimized laser parameters induces high antibody titers, which cannot be significantly increased by the tested adjuvants. Commercially available vaccines formulated without alum have the potential for successful TCI via laser-generated micropores, without the need for reformulation.

Background Specific immunotherapy via the subcutaneous or oral route is associated with local and, in some cases, systemic side effects and suffers from low patient compliance. Due to its unique immunological features, the skin represents a promising target tissue for effective and painless treatment of type I allergy. The current study was performed to compare the efficacy of transcutaneous immunotherapy via laser-generated micropores to subcutaneous injection. Methods BALB/c mice were sensitized by intraperitoneal injection of recombinant grass pollen allergen Phl p 5 together with alum. Subsequently, lung inflammation was induced by repeated intranasal challenge. During the treatment phase, adjuvant-free Phl p 5 was applied in solution to microporated skin or was subcutaneously injected. Lung function and cellular infiltration; Phl p 5–specific serum levels of IgG1, IgG2a, and IgE; and cytokine levels in bronchoalveolar lavage fluids as well as in supernatants of splenocyte cultures were assessed. Results Both therapeutic approaches reduced airway hyperresponsiveness and leukocyte infiltration into the lungs. Whereas subcutaneous immunotherapy induced a systemic increase in Th2-associated cytokine secretion, transcutaneous application revealed a general downregulation of Th1/Th2/Th17 responses. Successful therapy was associated with induction of IgG2a and an increase in FOXP3+ CD4+ T cells. Conclusions Transcutaneous immunotherapy via laser microporation is equally efficient compared with conventional subcutaneous treatment but avoids therapy-associated boosting of systemic Th2 immunity. Immunotherapy via laser-microporated skin combines a painless application route with the high efficacy known from subcutaneous injections and therefore represents a promising alternative to established forms of immunotherapy.

SummaryBackground Wheat (Triticum aestivum) is an important allergen source responsible for various clinical manifestations of allergy (i.e. food allergy, pollen allergy, respiratory allergy to flour-Baker's asthma). Objective The objective of this study was the molecular and immunological characterization of new recombinant wheat allergens and to evaluate their usefulness for the diagnosis of allergy to wheat. Methods A T. aestivum cDNA library was constructed and screened with serum IgE from patients suffering from wheat allergy to identify cDNAs coding for new wheat allergens. The allergen-encoding cDNAs were expressed in Escherichia coli and purified to homogeneity. IgE reactivity of recombinant proteins was analysed in RAST-based, non-denaturing dot blot experiments and by ELISA with sera from wheat allergic patients and their allergenic activity was assessed in basophil degranulation experiments. Results We report the molecular characterization, recombinant expression and purification of five wheat allergens, a thioredoxin h isoform, glutathione transferase, 1-Cys-peroxiredoxin, profilin and dehydrin. Homologous proteins were identified by sequence comparisons in various plants. 1-Cys-peroxiredoxin appeared to be the most relevant of the newly identified wheat allergens according to prevalence of IgE recognition and results from basophil degranulation experiments. It showed IgE cross-reactivity with seed proteins from barley, rye, rice, maize, soy, oat and spelt. 1-Cys-peroxiredoxin, glutathione transferase and dehydrin were mainly recognized by patients with baker's asthma but not wheat-induced food allergy. Conclusion and Clinical Relevance The characterized recombinant wheat allergens may be useful for the development of serological tests which allow the discrimination of different clinical manifestations of wheat allergy.

Background: Knowledge of allergen-specific T cell epitopes is a prerequisite not only for therapeutic approaches but also for elucidating immunological mechanisms of type I allergy. Ex vivo detection of allergen-specific T cells using class II tetramer technology has become an important tool for investigating immune responses in atopic and healthy individuals. Methods: Using 3H-thymidine incorporation assays, T cell epitopes specific for the major timothy grass pollen allergen Phl p 5.0101 were mapped in 11 allergic donors and two different mouse strains. Different protocols for expansion/restimulation of T cells from the blood of allergic donors and detection of allergen-specific T cells by Class II Ultimer staining were evaluated. Results: We identified several new Phl p 5.0101 class II T cell epitopes in allergic patients and confirmed previously published ones. Additionally, we discovered the major T cell epitopes in BALB/c and C57BL/6 mice. Using a novel Class II Ultimer, we detected epitope-specific T cells expanded from the blood of an allergic donor. Conclusions: Epitope mapping of Phl p 5.0101 revealed an immunodominant epitope in BALB/c and C57BL/6 mice and an immunodominant region in humans (amino acids 259–282), which was recognized by 8 out of 11 allergic donors. Detection of Phl p 5-specific T cells was demonstrated using a Class II Ultimer specific for epitope 196–210. Successful detection of ultimer-positive T cells was strongly dependent on a resting phase after in vitro expansion.

Background Tropomyosins represent clinically relevant seafood allergens but the role of mite tropomyosin, Der p 10, in house dust mite (HDM) allergy has not been studied in detail.

Cite this as: C. Madritsch, S. Flicker,S. Scheiblhofer, D. Zafred, T. Pavkov-Keller, J. Thalhamer, W. Keller and R. Valenta, Clinical & Experimental Allergy, 2011 (41) 270–280. Abstract Background Allergen recognition by IgE antibodies is a key event in allergic inflammation. Objective To construct a plasmid for the expression of human monoclonal IgE antibodies of any desired specificity and to express IgE specific for the major timothy grass pollen allergen Phl p 5. Methods In a first step, the DNA sequence coding for the IgG1 heavy chain was excised and replaced by the sequence coding for the human ɛ constant region gene in plasmid pLNOH2 expressing a human Phl p 5-specific IgG1 heavy chain. Then, this construct together with a second plasmid expressing the corresponding Phl p 5-specific light chain was co-expressed in COS-7 cells. The Phl p 5-specific IgE (rhuMabEP5) was analysed for allergen-specificity and isotype by ELISA. Cross-reactivity of rhuMabEP5 was investigated by immunoblotting using pollen extracts from various grass species. The allergenic activity of Phl p 5 was studied by exposing rat basophil leukaemia (RBL) cells expressing human-FcɛRI to rhuMabEP5 and Phl p 5. Results We report the construction of vector pLNOH2-P5IgE, for the expression of human IgE and exemplify its usefulness by the production of a complete and functional human monoclonal IgE (rhuMabEP5). rhuMabEP5 is specific for the grass pollen allergen Phl p 5 and cross-reacts with group 5 allergens in natural grass pollen extracts. RBL-release assays with rhuMabEP5 demonstrated that oligomerization does not contribute to the high allergenic activity of Phl p 5. Conclusion and Clinical Relevance Plasmid pLNOH2-P5IgE allowed the production of a fully functional human monoclonal IgE antibody specific for Phl p 5. Recombinant human IgE antibodies of defined specificity represent useful tools to investigate mechanisms underlying IgE-mediated allergies.

Purpose of review mRNA vaccines have recently been re-discovered as an attractive alternative to the more prominent DNA vaccines, as they harbor many advantages with respect to safety and regulatory issues. Whereas most mRNA vaccines are focused on tumor therapy, this type of vaccine has now also been successfully employed for prophylactic immunization against type I allergy in a mouse model. This concept differs from conventional immunotherapy in that it relies on immune deviation toward a TH1 phenotype, rather than induction of regulatory T cells or tolerance. Recent findings Conventional as well as self-replicating mRNA vaccines have demonstrated their potential to prevent the induction of an allergic phenotype in terms of allergen-specific IgE, allergy-associated cytokine profiles, eosinophilic lung infiltration, and airway hyperreactivity. Preliminary data raise the question whether TH1 immune deviation induced by mRNA vaccination resembles the natural phenotype of a certain proportion of nonatopic individuals. Recent findings Reservations regarding Good Manufacturing Practices manufacture costs, shelf life stability, and lack of immunogenicity due to rapid in-vivo degradation have been overcome by novel findings. Summary mRNA vaccines open the field for a safety-optimized prophylactic vaccination against allergic diseases. Future studies concerning long-term effects and vaccine-induced versus natural immune responses will be needed to transfer this knowledge to the clinics.

SummaryBackground Cow’s milk is one of the most common causes of food allergy affectingapproximately 2.5% of infants in the first years of their life. However, only limitedinformation regarding the allergenic activity of individual cow’s milk allergens is available.ObjectiveToanalysethefrequencyofIgEreactivityandtodeterminetheallergenicactivityofindividual cow’s milk allergens.MethodsA nitrocellulose-based microarray, based on purified natural and recombinant cow’smilk allergens was used to determine IgE reactivity profiles using sera from 78 cow’s milk-sensitized individuals of varying ages. The allergenic activity of the individual allergens wastested using patients’ sera for loading rat basophil leukaemia cells (RBL) expressing thea-chain of the human receptor FceRI.Results Using the microarray and the RBL assay, cow’s milk allergens were assessed forfrequency of IgE recognition and allergenic activity. Moreover, the RBL assay alloweddistinguishing individuals without or with mild clinical reactions from those with severesystemic or gastrointestinal symptoms as well as persons who grew out cow’s milk allergyfrom those who did not.Conclusions Component-resolved testing using milk allergen microarrays and RBL assaysseems to provide useful additional diagnostic information and may represent a basis forfuture forms of prophylactic and therapeutic strategies for cow’s milk allergy.Keywords Basophil degranulation, cow’s milk allergy, diagnosis, microarray, recombinantallergenSubmitted 15 March 2010; revised 8 June 2010; accepted 13 July 2010

Die genetische Immunisierung, oder auch DNA-Immunisierung genannt, hat sich im letzten Jahrzehnt als wirksame Methode zur Induktion von anti-allergischen Immunreaktionen im Tiermodell bewahrt. Die Wirkung von DNA-Impfstoffen beruht auf der Rekrutierung von Allergen-spezifischen Th1- und CD8+-Zellen und dem Aufbau eines Th1-Zytokin-Milieus. Die Kombination beider Aspekte wirkt in einem protektiven Ansatz gegen die Entwicklung einer Th2-Immunreaktion und vermindert in einem therapeutischen Ansatz eine bereits bestehende allergische Reaktion. Genetische Vakzine offnen innovative Auswege aus den Problemen, die mit der Immunisierung durch Proteine verbunden sind. Es konnen sowohl das Kreuzvernetzen von bereits vorhandenen IgE-Antikorpern auf Mastzellen und Basophilen als auch die Induktion von neuen IgE-Antikorpern vermieden werden. Neben der Beschreibung der Wirkungsweise der genetischen Immunisierung wird im vorliegenden Ubersichtsartikel die Entwicklung von auf Sicherheit und Wirksamkeit optimierten DNA-Vakzinen zur Allergiebehandlung vorgestellt.

Gene gun immunization has been associated with the induction of a heterologous type of immune response characterized by a T(H)1-like immune reaction on the cellular level, i.e. generation of IFN-gamma secreting CD8(+) T-cells, yet a T(H)2 biased serology as indicated by high IgG1:IgG2a ratios and induction of IgE. Nevertheless, gene gun immunization using the model molecule beta-galactosidase has been argued to prevent IgE induction and to promote T(H)1 cells with respect to allergy DNA immunization. In our current study, we evaluated the potential of gene gun immunization to prevent type I allergic reactions comparing beta-galactosidase with two clinically relevant allergens, and further investigated the effect of gene gun immunization on relevant lung parameters. BALB/c mice were immunized with plasmids encoding the birch pollen allergen Bet v 1, the grass pollen allergen Phl p 5, or the model molecule beta-galactosidase, either by gene gun or intradermal injection followed by sensitization and intranasal provocation with the respective allergen. IgG1 and IgG2a antibody titers were determined by ELISA. IgE levels were evaluated in a rat basophil release assay. The severity of eosinophilia was determined in bronchoalveolar lavages, and the overall infiltrate was analyzed by histology on lung paraffin sections. Gene gun immunization induced a T(H)2-biased immune reaction, which did not prevent from production of IgE after subsequent sensitization. This T(H)2 effect was influenced by the nature of the antigen, with a more pronounced T(H)2-bias for the allergens Bet v 1 and Phl p 5 compared to beta-galactosidase. Gene gun immunization with all three antigens promoted eosinophil influx into the lung and did not alleviate lung pathology after intranasal provocation. In contrast to needle injection of plasmid DNA, which triggers a clearly T(H)1-biased and allergy-preventing immune response, gene gun application fails to induce anti-allergic reactions with all tested antigens and is therefore contraindicated for allergen-specific immunotherapy.

Experimental leishmaniasis represents a suitable model to analyze Th1-type associated immunity. In C57BL/6 mice healing of leishmaniasis correlates with activation of Th1 cells. Recently, it could be demonstrated that dermal dendritic cells rather than epidermal Langerhans cells are responsible for the activation of Th1 cells after infection, indicating a necessary reconsideration of the role of Langerhans cells. In our current work, epidermal application of Leishmania-antigen prior to infection resulted in an atypical course of disease that is characterized by an impaired Leishmania-specific Th1 response. Consequently, these mice cannot manage an efficient elimination of the parasites at the site of infection. These data point to the activation of immunomodulatory effects by epidermal incorporation of antigen.

Malaria is the world's major parasitic disease, for which effective control measures are urgently needed. Despite considerable efforts, no successful vaccine against malaria has been developed so far. The method of DNA-based immunization offers the possibility to induce both antibody- and cell-mediated immune responses to a variety of antigens. The flexibility of the DNA vaccine technology permits the combination of several antigens from different developmental stages of the parasite's complicated life cycle. This review covers the development of DNA-based immunization against malaria from initial experiments in small animals to recently conducted clinical studies. Focusing on one of the best characterized malaria vaccine candidate antigens, the circumsporozoite protein, an overview of strategies to enhance vaccine efficacy is provided. Advanced application methods such as the gene gun technology or the needle-less jet injection device are described. As DNA vaccination represents a relatively new methodology, safety concerns associated with planned clinical applications are discussed. In summary, this novel type of vaccine has to be considered as a promising tool for future malaria vaccination strategies.

Background: Allergic diseases have become a major public health problem in developed countries; yet, no reliable, safe and consistently effective treatment is available. DNA immunization has been shown to prevent and balance established allergic responses, however, the high dose of conventional DNA vaccines necessary for the induction of anti-allergic reactions and their poor immunogenicity in primates require the development of new allergy DNA vaccines. We evaluated protective and therapeutic effects of a Semliki-Forest Virus replicase-based vs a conventional DNA vaccine in BALB/c mice using the model allergen β-galactosidase. Methods: Immunoglobulin (Ig)E suppression was determined by a basophil release assay as an in vitro correlate for allergen-specific crosslinking capacity of IgE reflecting the in vivo situation in an allergic individual. Th1 memory responses were measured by cytokine detection via enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot assay (ELISPOT). Results: Nanogram amounts of a replicase-based vector triggered a Th1 response comparable with that achieved with the injection of 20 000-times more copies of a conventional DNA plasmid, and induced IgE suppression in both a protective and a therapeutic setting. Conclusions: Replicase-based DNA vaccines fulfill the stringent criteria for an allergy DNA vaccine, i.e. low dose, strong Th1 immunogenicity and memory, lack of ‘therapy-induced’ IgE production and anaphylactic side effects. Moreover, by triggering apoptosis in transfected cells, their unique ‘immunize and disappear’ feature minimizes the hypothetical risks of genomic integration or induction of autoimmunity.

Background Hypoallergenic immunotherapy of type I allergies aims at inducing T-cell immunity while avoiding cross-linking of pre-existing IgE. DNA-based immunotherapy depends on the recruitment of antigen-specific T H 1 cells and therefore has to provide the whole repertoire of T-cell epitopes. Ubiquitination offers a general approach for the production of hypoallergenic DNA vaccines. Objective A DNA-based vaccine encoding the major birch pollen allergen Bet v 1 stably linked to ubiquitin was evaluated for its antiallergic potential in a BALB/c mouse model of allergy. Methods Plasmid DNA was applied to mice before (preventive) or after (therapeutic) sensitization with recombinant Bet v 1. In the preventive setting, mice were exposed to aerosolized allergen in addition. Cytokine production was monitored via ELISPOT and Luminex. IgG 1 , IgG 2a , and IgE subclass antibody titers were determined by ELISA. In vitro antigen-specific cross-linking of IgE was measured in a degranulation assay. Bronchoalveolar lavages were analyzed for leukocyte subsets as well as for IFN-γ and IL-5, and paraffin sections of lungs were examined for mucus production and endothelial damage. Results Prevaccination with ubiquitinated Bet v 1–stimulated T H 1-biased immune responses with concomitant suppression of functional IgE, reduction of eosinophil counts in bronchoalveolar lavages, and alleviation of lung pathology, and could also suppress an ongoing IgE response in a therapeutic setting. Conclusion The data clearly demonstrate that hypoallergenic DNA vaccines encoding ubiquitin fusion constructs induce effective antiallergic immune responses. Clinical implications Ubiquitination of allergen gene vaccines eliminates the risk of IgE cross-linking, thereby meeting the safety requirements for clinical applications.

Genetic immunization has proven a powerful method to induce antiallergic immune responses. The underlying functional principle has been described to be based on the recruitment of allergen-specific Th1 cells, CD8+ cells and the establishment of a Th1 cytokine milieu, which prevent the development of a Th2-biased response in a protective setup and can balance an ongoing Th2-type response in a therapeutic situation. Genetic immunization with plasmid DNA offers innovative solutions to the major problems associated with protein immunization, such as crosslinking of pre-existing immunoglobulin E on mast cells/basophils or induction of de novo synthesis of immunoglobulin E by the protein immunization itself. It easily enables the routine production of hypoallergenic vaccines, which do not translate native allergens, thus avoiding potential anaphylactic side effects. DNA vaccines can also be applied as mixtures of single vaccines, making them interesting candidates for treatment based on component-resolved diagnosis, followed by an individualized therapy with the relevant allergens. In addition to the description of up-to-date allergen gene vaccine approaches, this review gives an overview of animal studies dealing with the following topics: danger signals as the inherent adjuvant properties, methods to optimize the vaccine immunogenicity, modulation of the immune response, nonparenteral applications and low-dose vaccination strategies.

The immunogenicity of recombinant protein or anti-viral DNA vaccines can be significantly improved by the addition of tandem copies of the complement fragment C3d. We sought to determine if the efficacy of a circumsporozoite protein (CSP)-based DNA vaccine delivered to mouse skin by gene gun was improved by using this strategy. Instead, we found that C3d suppressed the protective immunity against Plasmodium berghei malaria infection and deviated immunity, most notably by suppressing the induction of antibodies specific for the CSP C-terminal flanking sequence and by suppressing the induction of CSP-specific IL-4-producing spleen cells. We further showed that C3d bound to the C-terminal flanking sequence of the CSP, which may explain the immune deviation observed in CS/C3d chimeric antigen. We have thus identified C3d-mediated epitope masking and shifting of both the humoral and cellular immune responses as a potential novel escape mechanism, which plasmodia may use to divert the induction of protective immunity.

The outer surface protein C (OspC) of Borrelia burgdorferi , the spirochete that causes Lyme disease, is a promising candidate for a vaccine against borreliosis. BALB/c and C3H/HeJ mice were immunized either with recombinant OspC protein or with plasmid DNA encoding OspC fused to the human tissue plasminogen activator leader sequence (pCMV-TPA/ZS7). The influence of the route of administering the DNA and the use of oligodeoxynucleotides containing CpG-motifs on the development of the immune response was investigated. In both mouse strains, protein as well as gene-gun immunization induced Th2 type responses, whereas needle injection of plasmid DNA resulted in Th1 type antibody production. Co-injection of CpG-motifs did not significantly modify the response type in any immunization group, as indicated by only marginal changes of antibody subclass distribution. The protection rate after challenge with 10 4 B. burgdorferi organisms per mouse was between 80% and 100% for all groups. These results demonstrate, for the first time, that a DNA vaccine encoding OspC of B. burgdorferi is suitable for inducing protection against Lyme borreliosis.

The mode of administering a DNA vaccine can influence the type of immune response induced by the vaccine. For instance, application of a DNA vaccine by gene gun typically induces a Th2-type reaction, whereas needle inoculation triggers a Th1 response. It has been proposed that the approximately 100-fold difference in the amount of DNA administered by these two methods is the critical factor determining whether a Th1 or a Th2 response is made. To test this hypothesis, BALB/c mice were immunized with two plasmid DNA constructs encoding different proteins (OspC/ZS7 of Borrelia burgdorferi and Bet v 1a, the major birch pollen allergen). Both vaccines were applied by needle and/or by gene gun immunization at the same and at different sites of injection. An analysis of the IgG subclass distribution and measurement of IFN-gamma after antigen-specific lymphoproliferation does not support the widely accepted view that Th2-type immunity induced by gene gun application is solely due to the low amount of injected plasmid DNA thus falling below the critical concentration of CpG motifs necessary for Th1-induction. Furthermore, the data also indicate a strong and even systemic adjuvant effect of the gene gun shot itself.

Capsule summary Naturally acquired immunity against allergens utilizes a broad spectrum of response types besides T regulatory cells. Therapeutic concepts should not be limited to T regulatory cell induction, but take advantage of the diversity found in natural responses.

DNA vaccines represent a novel approach for protective and therapeutic intervention against type I allergies. In contrast to classical subcutaneous immunotherapy, which relies on the injection of alum-adsorbed protein extracts, DNA vaccines do not suffer from side effects such as anaphylaxis or therapy-induced IgE antibodies. In animal models, DNA vaccines have been demonstrated to prevent TH2 sensitization or balance an existing TH2-mediated allergic immune response by induction of TH1 or regulatory T cells, rendering them promising candidates for prophylactic vaccination as well as therapy. In this chapter, we discuss methods relevant for evaluation of DNA expression vectors for targeting antigen to different cellular compartments for use as a vaccine in an asthma mouse model. Attaching signal sequences has proven to be a successful way to manipulate and boost the immune responses following DNA immunization and also creating hypoallergenic DNA vaccines.

The C terminus of the circumsporozoite protein (CSP) is anchored to the parasite cell membrane by a glycosylphosphatidylinositol (GPI) glycolipid. This GPI signal sequence functions poorly in heterologous eukaryotic cells, causing CSP retention within internal cell organelles during genetic immunization. Cellular location of antigen has quantitative and qualitative effects on immune responses induced by genetic immunization. Removal of the GPI signal sequence had a profound effect on induction and efficacy of CSP-specific immune response after genetic immunization of BALB/c mice with a gene gun. The CSP produced from the plasmid lacking the GPI anchor signal sequence (CSP-A) was secreted and soluble, but that produced by the CSP+A plasmid was not. The CSP-A plasmid induced a highly polarized Th2 type response, in which the CSP-specific IgG antibody titer was three- to fourfold higher, and the protective effect was significantly greater than that induced by the CSP+A plasmid. Thus, these two physical forms of CSP induced quantitatively and qualitatively different immune responses that also differed in protective efficacy. Engineering plasmid constructs for proper cellular localization of gene products is a primary consideration for the preparation of optimally efficacious DNA vaccines.

The circumsporozoite protein (CSP) from the surface of sporozoite stagePlasmodiumsp. malaria parasites is among the most important of the malaria vaccine candidates. Gene gun injection of genetic vaccines encodingPlasmodium bergheiCSP induces a significant protective effect against sporozoite challenge; however, intramuscular injection does not. In the present study we compared the immune responses and protective effects induced byP. bergheiCSP genetic vaccines delivered intradermally with a needle or epidermally with a gene gun. Mice were immunized three times at 4-week intervals and challenged by a single infectious mosquito bite. Although 50 times more DNA was administered by needle than by gene gun, the latter method induced significantly greater protection against infection. Intradermal injection of the CSP genetic vaccine induced a strong Th1-type immune response characterized by a dominant CSP-specific immunoglobulin G2a (IgG2a) humoral response and high levels of gamma interferon produced by splenic T cells. Gene gun injection induced a predominantly Th2-type immune response characterized by a high IgG1/IgG2a ratio and significant IgE production. Neither method generated measurable cytotoxic T lymphocyte activity. The results indicate that a gene gun-mediated CS-specific Th2-type response may be best for protecting against malarial sporozoite infection when the route of parasite entry is via mosquito bite.

Nuclear retinoid receptors - retinoic acid inducible transcription factors - participate in pathways influencing many components of the immune system. In the present study in vivo effects of DNA-based immunization of mice on binding parameters of all-trans retinoic acid receptors (RARs) in spleen cell nuclei was investigated. A eucaryotic expression vector encoding the gene for the model enzyme beta-galactosidase of Escherichia coli (pCMV-beta) was used for intradermal injection. Furthermore, immunostimulatory CpG motifs, which stimulate the expression of various cytokines and may serve as a 'danger signal' for the mammalian immune system, were coinjected as oligodeoxynucleotides. The results demonstrate that the concentration of RARs was significantly reduced in the late phase of the primary immune response (21 days after injection of plasmid DNA-indicated by high affinity IgG antibodies and IFN-gamma expression). Coinjection of CpG motifs did not change the course of the humoral response but enhanced and accelerated the proliferative response and expression of IFN-gamma, which correlated with the reduced RARs concentration.

Purpose of review This review summarizes recent preclinical and human studies evaluating allergen-specific immunotherapy via the transcutaneous route, and provides a rationale for the application of modified allergens with reduced allergenicity. Furthermore, it covers approaches to generate hypoallergenic conjugates for specific dendritic cell targeting. Recent findings Efficacy and safety of specific immunotherapy by application of allergens to the skin have been demonstrated in both animal models as well as clinical trials. However, localized adverse events have been reported, and delivery of antigens via barrier-disrupted skin has been linked to the induction of unwanted T helper 2-biased immune responses and allergic sensitization. Coupling of carbohydrates to allergens has been shown to induce formation of nanoparticles, which can specifically target dendritic cells and potentiate immune responses, and by masking B-cell epitopes, can render the molecules hypoallergenic. Summary Due to its abundance of immunocompetent cells, the skin represents an attractive target tissue for novel and enhanced immunotherapeutic approaches. However, in order to avoid adverse events and therapy-induced sensitizations, transcutaneous immunotherapy requires the use of formulations with reduced allergenic potential. Combining novel hypoallergenic conjugates with painless transcutaneous immunization techniques may provide an efficient and patient-friendly alternative to the standard specific immunotherapy practices.

Today, type I allergies affect more than 30% of the population in western industrialized countries posing an increasing burden on public health systems. In this book chapter we provide an overview on the molecular characteristics of allergens and the mechanisms of allergic sensitization. Risk factors for sensitization such as genetic predisposition or environmental factors (“hygiene hypothesis”) are discussed and the current standards of diagnosis and medication are summarized. As classical allergen-specific immunotherapy suffers from unwanted side-effects, low patient compliance as well as insufficient efficacy, this chapter focuses on novel therapeutic approaches to overcome these limitations. These include new molecules, such as recombinant (hypo-) allergens or peptides but also advanced vector vaccines, and genetic vaccines. Such vaccine types address specific receptors of the innate immune system, resulting in increased immunogenicity and modulation of unwanted TH2 type responses. Finally, alternative routes to the standard subcutaneous injection or sublingual application are presented, which target highly immunocompetent tissues such as the skin or the lymph nodes.

The gene gun technology can not only be employed for efficient transfer of gene vaccines into upper layers of the skin, but also for application of protein antigens. As a tissue rich in professional antigen presenting cells, the skin represents an attractive target for immunizations. In this chapter we present a method for delivery of the model antigen ovalbumin into the skin of mice termed epidermal antigen incorporation and describe in detail how antigen-specific proliferation in draining lymph nodes can be followed by flow cytometry.

The gene gun technology can not only be employed for efficient transfer of gene vaccines into upper layers of the skin, but also for application of protein antigens. As a tissue rich in professional antigen presenting cells, the skin represents an attractive target for immunizations. In this chapter we present a method for delivery of the model antigen ovalbumin into the skin of mice termed epidermal antigen incorporation and describe in detail how antigen-specific proliferation in draining lymph nodes can be followed by flow cytometry.

Wheat is an essential element in our nutrition but one of the most important food allergen sources. Wheat allergic patients often suffer from severe gastrointestinal and systemic allergic reactions after wheat ingestion. In this study, we report the molecular and immunological characterization of a new major wheat food allergen, Tri a 36. The cDNA coding for a C-terminal fragment of Tri a 36 was isolated by screening a wheat seed cDNA expression library with serum IgE from wheat food-allergic patients. Tri a 36 is a 369-aa protein with a hydrophobic 25-aa N-terminal leader peptide. According to sequence comparison it belongs to the low m.w. glutenin subunits, which can be found in a variety of cereals. The mature allergen contains an N-terminal domain, a repetitive domain that is rich in glutamine and proline residues, and three C-terminal domains with eight cysteine residues contributing to intra- and intermolecular disulfide bonds. Recombinant Tri a 36 was expressed in Escherichia coli and purified as soluble protein. It reacted with IgE Abs of ∼80% of wheat food-allergic patients, showed IgE cross-reactivity with related allergens in rye, barley, oat, spelt, and rice, and induced specific and dose-dependent basophil activation. Even after extensive in vitro gastric and duodenal digestion, Tri a 36 released distinct IgE-reactive fragments and was highly resistant against boiling. Thus, recombinant Tri a 36 is a major wheat food allergen that can be used for the molecular diagnosis of, and for the development of specific immunotherapy strategies against, wheat food allergy.

Allergic diseases are on the increase and current therapies are lacking in efficacy and patient compliance. In recent years, the idea of prophylactic measures, especially for children at high risk for allergy, has become increasingly popular. This review summarizes the available preclinical data for protective allergy vaccines, with a focus on one of the most promising vaccine candidates; mRNA vaccines. Recently, mRNA vaccines have been rediscovered as an alternative to their more prominent counterparts, the DNA vaccines. Allergen-encoding mRNA vaccines elicit long-lasting protection from sensitization, and induce a type of immunity similar to the natural protective response that is acquired in the presence of microbial burden early in life. Owing to their excellent safety profile, they represent the ideal candidates for a vaccine that aims to protect at-risk children who have not yet been sensitized to allergens.

The constant rise in allergies in Western industrialized countries has fueled efforts to develop novel therapies to treat the immunological cause of disease rather than merely ameliorating symptoms. Gene vaccines against allergic diseases may be an attractive alternative to classical specific immunotherapy (SIT) avoiding its pitfalls such as potential side effects as well as low patient compliance. While SIT is commonly believed to rely on the generation of regulatory immune reactions and blocking antibodies, gene based allergy vaccines rather exert immune deviation by balancing allergic TH2 reactions through the generation of allergen specific TH1 cells. In contrast to SIT, gene based allergy vaccines may also have a high potential for prophylactic applications. Already small amounts of translated allergen in the context of DNA/RNA vaccine-inherent danger signals prime a milieu that counteracts the induction of allergic immune responses. Such prophylactic vaccines may very well resemble naturally acquired protection from allergy as it is observed in individuals exposed to microbial compounds during early childhood (hygiene hypothesis). In this chapter, we will discuss different types of DNA vaccines that have been designed to prevent from allergy, and the potential safety hazards associated with their application for prophylactic vaccination of children. Furthermore, we will demonstrate that RNA vaccines are as effective as DNA vaccines, yet with a much higher safety profile, thus making them the most promising candidates for prophylactic vaccination against allergic diseases.

Gene vaccines have emerged as attractive alternative to conventional vaccines as they have the potential to activate both branches of the immune system, similar to live attenuated vaccines, but at a higher safety profile and with superior efficacy in production and costs. After impressive performance in small animal models, gene vaccines were quickly transferred into the clinics without detailed knowledge of the underlying molecular and immunological mechanisms. As a result, these early clinical trials led to disappointing results, nearly terminating the “DNA vaccine revolution.” Today, we know a lot more about vector design, uptake and processing of gene vaccines, immunostimulatory properties of DNA and RNA, and about numerous ways to modulate immune responses. Utilizing this knowledge has led to a new generation of improved vaccines and delivery modalities, and progress in the clinics is emerging. This introductory chapter will give a broad overview on the current status of gene vaccine design, their delivery and fate after introduction into the body, their immunostimulatory capacities, and finally, the processing and presentation of vaccine-encoded antigen to the immune system.

The complement fragment C3d mediates B-cell activation via simultaneous engagement of the B-cell receptor and CD21 by antigen/C3d conjugates. Several studies demonstrated the potential of C3d as a molecular adjuvant for vaccination. In this work, C3d exerted differential effects on humoral immune responses after gene gun immunization of mice with plasmids encoding the malaria blood stage antigen MSP1(42) depending on the nature of the protein (Plasmodium falciparum vs. Plasmodium berghei MSP), the localization of the C3d moiety (C-terminal vs. N-terminal), and the presence of putative N-glycosylation sites. No improvement of protective efficacy by C3d attachment or mutation of glycosylation sites could be demonstrated by in vitro parasite growth inhibition assays or in vivo blood stage parasite challenges. Our data underscore the controversial role of C3d as molecular adjuvant.