An immunoinformatics approach for identification and characterization of T cell-mediated heterologous immunity between RNA viruses and allergens

Asthma is one of the major chronic inflammatory disorders worldwide. Respiratory virus infections and exposure to allergens are important risk factors for asthma development. Our group previously demonstrated an influenza-mediated protective effect over experimental asthma in a murine model, which was dependent on crossreactive T effector memory cells. The hypothesis of this research work was that virus- mediated heterologous immune response is a broadly applicable concept for several respiratory viruses and environmental allergens. This immune response may be mediated by cross-reactive virus-specific memory T cells, which react in a T1-driven immune response upon allergen exposure. In order to test this hypothesis, a comprehensive in-silico pipeline for prediction of potentially cross-reactive T cell epitope pairs between several respiratory viruses and allergens was developed, taking MHC binding affinity and sequence similarity of the T cell epitopes into consideration. An additional scoring system further characterized and prioritized the allergen counterpart based on clinical relevance and conservation criteria. Mouse Balb/c MHC class I epitope pairs were validated for MHC stabilization in an in-vitro assay and binder pairs were used for further ex-vivo and in-vivo experiments. Using an RSV A2 infection model, ex-vivo T cell stimulation assays of lung cells and splenocytes confirmed immunogenicity of the predicted virus peptides as well as cross-reactivity of the corresponding allergen peptides. Additionally, dual pentamer staining of lung cells of RSV-infected mice with the predicted candidate pair RSV A2 L356-364 FYNSMLNNI/Asp f 4192-200 WYGNSALTI revealed virus- and allergen- specific CD8+ T effector memory cells, as well as double positive T cells. Based on this result and the fact that several predicted candidate pairs derived from Aspergillus fumigatus (Asp f), mice were immunized with a pool of RSV A2-derived peptides that were predicted to cross-react with Asp f-derived peptides. Ex-vivo stimulation of splenocytes with the predicted Asp f-derived peptides resulted in increased proliferation, activation and cytokine production of CD8+ T cells. Subsequently, a potentially protective effect of RSV A2 infection towards development of Asp f-induced experimental asthma was investigated. A preceding RSV infection resulted in less eosinophils and T2 cytokines in the BAL of allergic asthmatic animals compared to those without virus infection, as well as a trend for reduced abundance neutrophils and T2-biased CD8+ T cells in the lung. Additionally, lung histology sections confirmed less mucus-producing goblet cells and inflammation. In order to investigate the contribution of the predicted cross-reactive T cell epitopes in the attenuation of the allergic asthmatic response, mice were immunized with the predicted RSV-derived peptides and subsequently subjected to the Asp fmouse model. These animals had lower numbers of eosinophils in the BAL, displayed a trend for reduced IL5+ CD8+ T cells and increased abundance of IFNγ+ CD8+ T cells in the lung compared to allergic asthmatic mice without prior peptide immunization. In addition to RSV A2, candidate epitope pairs for the virus strains of the seasonal quadrivalent influenza vaccine 2019/2020 (QIV) were predicted and validated invitro. A mouse model of influenza vaccination was established, inducing both humoral response and virus-specific T cell response. Mice were vaccinated with the QIV and ex-vivo stimulation of splenocytes with the predicted allergen peptides resulted in increased induction of activated CD8+ CD69+ T cells compared to control stimulation and mock animals. Subsequently, QIV immunization was combined with an HDM-induced experimental asthma model and, similarly to the RSV/Asp f model, hallmarks of allergic asthma were attenuated, including numbers of eosinophils, lung inflammation and mucus production. In summary, these data confirm virus-induced heterologous immune responses towards environmental allergens, which result in attenuation of allergen-mediated experimental asthma. Several epidemiologically relevant respiratory RNA viruses play a role in this regard. A more extensive virus peptide pool may be needed to induce the same degree of attenuation with peptide immunization versus virus infection. Further experiments using human biomaterial for investigation of cross-reactive T cell populations among asthmatic and healthy individuals who have or have not been vaccinated against influenza or following specific virus infections would support the translational potential of our findings. Evidence in this regard will have important implications for future peptide vaccination strategies, inducing dual antiviral and anti-allergic potential.