Lactobacillus rhamnosus GG expressing birch pollen proteins for allergen-specific immunotherapy of experimental asthma in mice.

Birch pollen allergy is a common cause of allergic airway disease that affects approximately 100 million people worldwide, presenting a compelling economic and social burden. The only potentially curative allergy treatment is allergen-specific immunotherapy, which involves administration of allergens to the patient in order to induce long-term tolerance. However, in its current form allergen-specific immunotherapy is linked to a number of drawbacks, including variable efficacy, inconvenient administration routes, high costs and potentially life-threatening side effects. A promising alternative strategy for the administration of therapeutic molecules involves their expression in probiotic bacteria, which are then implemented as live mucosal delivery vehicles. Model probiotic strains, such as Lactobacillus rhamnosus GG and Lactobacillus rhamnosus GR-1, are especially interesting in the treatment of allergic asthma due to their intrinsic immunomodulatory and immunostimulatory effects. These qualities of the probiotic strains, in combination with their proposed ability to produce and deliver allergens to mucosal surfaces, could facilitate effective administration of therapeutic molecules in the context of allergic asthma and other diseases. Therefore, the aim of this PhD project was to investigate the effects of genetically engineered and wild type L. rhamnosus GG and L. rhamnosus GR-1 as preventive treatment of experimental allergic asthma. Recombinant L. rhamnosus strains were engineered to express the major birch pollen allergen Bet v 1, to which 90-95 % of birch pollen-allergic patients are sensitized. To test the efficacy of these probiotic strains, several cost-effective and easy-to-implement mouse models with typical hallmarks reminiscent of birch pollen-induced allergic asthma were developed. For allergic sensitization of mice, biologically active Bet v 1 was produced and purified in E. coli BL21 (DE3). Induction of allergic airway disease by the intranasal route could be achieved with either the whole birch pollen extract containing Bet v 1, or the combination of Bet v 1 with Staphylococcus aureus enterotoxin B. Bet v 1 thus requires an adjuvant for intranasal allergic sensitization.

In the studied mouse models of birch pollen-induced allergic asthma, significant benefits of both intragastric and intranasal administration of L. rhamnosus strains could be demonstrated. An improvement of airway hyperreactivity by one or several L. rhamnosus strains in each experimental set-up was observed. This represents an important outcome with potential clinical implications, as airway hyperreactivity is a defining pathophysiological hallmark of asthma in humans. Interestingly, this was not always linked to significant changes in allergic sensitization or airway inflammation. Intranasal administration of recombinant L. rhamnosus strains producing Bet v 1 elicited strong systemic Bet v 1-specific antibody responses, which did not occur after intragastric administration. The most significant effect on allergic asthma development was likewise obtained with intranasal, but not intragastric, application of wild type L. rhamnosus GG, which improved both airway hyperreactivity and airway inflammation. Wild type L. rhamnosus GR-1 strain improved airway function in a therapeutic, but not in a preventive setting of intranasal treatment of experimental allergic asthma. These observations suggest that intranasal probiotic instillations could provide an effective alternative to the traditional oral route.

Heterogeniety in the mouse responses towards pretreatment with the wild type and recombinant L. rhamnosus strains was clearly observed, which was most prominent when the strains were administered intragastrically. Variation of gut microbiome composition among individual mice was likewise detected. A correlation was demonstrated between an increased shift in microbiome composition after asthma induction and excerbation of airway hyperreactivity, allergic inflammation, and allergic sensitization. The observed differences in gut microbiota and their link with the severity of allergic airway disease suggest that this host parameter has to be taken into account in probiotic study outcomes.

Taken together, these results highlight the significant potential of recombinant and wild type probiotic strains in prevention and treatment of allergic asthma. The observed consistent impact of probiotic intervention on airway hyperreactivity, which is the most prominent feature of asthma in humans, is an especially promising observation. Further unraveling of the in vivo outcomes of probiotic administration in mouse models and understanding of the factors that influence their beneficial effects in the host will promote optimal clinical application of both recombinant and wild type strains.