For the design of potent subunit vaccines, it is of paramount importance to identify all antigens immunologically recognized by a patient population infected with a pathogen. We have developed a rapid and efficient procedure to identify such commonly recognized antigens, and here we provide a comprehensive in vivo antigenic profile of Staphylococcus aureus, an important human pathogen. S. aureus peptides were displayed on the surface of Escherichia coli via fusion to one of two outer membrane proteins (LamB and FhuA) and probed with sera selected for high Ab titer and opsonic activity. A total of 60 antigenic proteins were identified, most of which are located or predicted to be located on the surface of the bacterium or secreted. The identification of these antigens and their reactivity with individual sera from patients and healthy individuals greatly facilitate the selection of promising vaccine candidates for further evaluation. This approach, which makes use of whole genome sequence information, has the potential to greatly accelerate and facilitate the formulation of novel vaccines and is applicable to any pathogen that induces Abs in humans and͞or experimental animals.

Background Several studies have demonstrated that mucosal administration of soluble antigens can prevent the onset or reduce the severity of certain autoimmune diseases or allergies. Few studies exist showing the ef®cacy of mucosal tolerance for therapy of such diseases. Objective The aim of the present study was to modulate an allergic immune response by intranasal antigen administration in an already sensitized organism. Methods A murine model of allergic asthma to birch pollen (BP) and its major allergen Bet v 1 was utilized. Sensitized mice were intranasally treated with recombinant (r)Bet v 1 in different concentrations and at different intervals. On the day the mice were killed, blood and bronchoalveolar lavagē uids were taken and immediate type I skin tests were performed. T cell proliferation and cytokine production (interleukin (IL)-5, interferon (IFN)-g) were measured in spleen and lung cell cultures.Results Mucosal treatment with rBet v 1 (3 Â 50 mg in 4 day intervals) led to a reduction of type I skin reactions, suppressed immunoglobulin (Ig)G1/IgE antibody levels and markedly decreased IL-5 and IFN-g production in vitro in spleen and lung cell cultures. Moreover, lung in¯ammation (i.e. eosinophilia and IL-5 levels in bronchoalveolar lavage¯uids) was signi®cantly suppressed by the treatment. Conclusion Our results demonstrate that intranasal treatment with rBet v 1 reduced systemic allergic immune responses as well as airway in¯ammation in BP-sensitized mice. We therefore suggest that mucosal tolerance induction with recombinant allergens could be a promising concept for the therapy of allergic diseases.

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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.

SUMMARYUp to 5% of the population suffer from systemic, 19% from local allergic hypersensitivity reactions to stinging insects. Even though speci®c immunotherapy is very effective in treating allergy to insect venom, new concepts of treatment strategies with only the disease eliciting allergen in recombinant form, along with antigen application via a less invasive route might be suggested for enhanced treatment ef®cacy and compliance. In the present study we aimed (i) to establish a mouse model of wasp venom allergy, mimicking the natural mode of sensitization, and (ii) to develop a prophylactic treatment strategy based on mucosal tolerance induction, using one major wasp venom allergen in recombinant form, i.e. recombinant (r)Ves v 5. Immunization with wasp venom ± with or without the use of the adjuvant aluminium hydroxide ± led to comparable T helper 2-like immune responses in vivo and in vitro. Intranasal administration of rVes v 5 prior to sensitization with wasp venom resulted in a signi®cant reduction of wasp venom-speci®c antibody levels (immunoglobulin E (IgE)/ IgG2a), type I hypersensitivity reactions in vivo and cytokine production in vitro. Pretreatment with the whole venom was less effective and caused toxic side reactions in higher concentrations, suggesting a favourable use of the recombinant venom allergen for mucosal application. Increased mRNA levels of transforming growth factor-b and interleukin-10, along with adoptive cell transfer experiments indicated that the immunosuppression after intranasal rVes v 5-application has been mediated by regulatory mechanisms. This is further supported by the fact that the immunosuppression to rVes v 5 was associated with a bystander suppression to the unrelated aero-allergen Bet v 1. In conclusion, we demonstrated that the intranasal application of recombinant Ves v 5 prevented subsequent allergic sensitization to all components of the whole wasp venom. As allergy to insect venom develops in dependence of the frequency of insect stings, a prophylactic treatment based on mucosal tolerance induction with recombinant allergens might be of interest for people at high risk to frequent exposure to the stinging insects.

SUMMARYThe pathophysiological and immunological characteristics of allergic immune responses are controlled by a variety of factors. We have studied the extent to which the route of sensitization influences allergenspecific IgE synthesis and local airway inflammation using a mouse model of allergic sensitization to the major birch pollen allergen Bet v 1. Sensitization of BALB/c mice with recombinant (r)Bet v 1 was performed using intraperitoneal (IP), subcutaneous (SC) or aerosol (AS) sensitization protocols. Mice were analysed for allergen-specific serum antibodies by ELISA and IgE-dependent basophil degranulation. Proliferative responses and cytokine production of splenocytes were measured upon Bet v 1 stimulation in vitro . Bronchoalveolar lavages were performed after airway challenge with aerosolized birch pollen extract for assessment of eosinophilic airway inflammation and local cytokine production in vivo. Highest allergen specific IgE levels and IgE-dependent basophil degranulation were achieved using the SC route. High IL-5 production by spleen and lung cells was associated with pronounced eosinophilia in bronchoalveolar lavages. After IP sensitization, despite giving the highest IgG levels, only low IgE levels, basophil degranulation and IL-5 production were seen. On the other hand, AS sensitization, resulting in the lowest systemic IgE and IL-5 levels, led to a comparably strong airway inflammation as the SC route. Our finding that the route of sensitization can result in a dissociation of local and systemic immune responses may contribute to a better understanding of the pathogenesis of allergic diseases and help to develop new treatment strategies.Keywords type I allergy murine model sensitization route local immune response systemic immune response

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