DETECTING ALLERGEN-SPECIFIC B CELLS IN MURINE MODELS OF NOVEL PEPTIDE IMMUNOTHERAPY

Abstract

The prevalence of allergic disease is increasing, with 10-20% of adults globally classified as allergic. Domestic cats are a common source of allergic sensitization, with over 90% of cat allergic individuals being immunoglobulin (Ig) E sensitized to the Felis domesticus (Fel d) 1 protein. During an allergic reaction, affected individuals can experience a wide range of symptoms some of which are life-threatening. Current treatments for cat allergies fail to address the underlying causes of the disease. Peptide immunotherapy using T cell epitopes has demonstrated a strong safety profile and resulted in the development of long-lasting clinical tolerance in studies involving allergic human subjects. However, stability issues, varying responses and manufacturing cost were downsides to the therapy. mRNA peptide immunotherapy vaccines, which encoded the T cell epitopes were a proposed solution. This project aimed to produce and validate Fel d 1 B cell tetramers and to evaluate B cell responses in both naïve and sensitized mice immunized with two prototype vaccines (VXL01, VXL02). Fel d 1 B cell tetramers were produced, tested to ensure specificity and optimized for use in flow cytometry. Splenocytes from mice immunized with vaccine only, immunized with vaccine and given whole-allergen exposure, or allergically sensitized with Fel d 1 and immunized with vaccine were stained and analyzed. Fel d 1 specific B cells were not detected in the vaccine immunized only groups. Neither prototype peptide immunotherapy mRNA vaccine reduced allergic responses in Fel d 1 sensitized mice. There were no statistically significant changes in BAL cellularity, serum IgG and IgE when compared to control at study-endpoint. Fel d 1 sensitized mice immunized with VXL02 had a significantly higher percentage of Fel d 1 tetramer+ B cells than the vehicle alone group, suggesting the vaccine may be capable of activating an established memory B cell pool. In conclusion, performing tetramer analysis on splenocyte populations was both sensitive and antigen-specific. Immunization with prototype vaccines alone did not activate a naïve Fel d 1 specific B cell response but appeared to expand Fel d 1 specific memory B cells. Fel d 1 specific B cells could be detected in mice sensitized to Fel d 1 prior to immunization with the mRNA vaccines with no changes in markers of allergic disease between vaccinated and control groups, indicating the vaccines were not effective at reducing the Th2 allergic disease phenotype.