Isolation of Dengue neutralizing antibodies from single cell sorted human antigen specific memory B cell cultures (VAC11P.1107)

Abstract

Abstract Monitoring antigen-specific memory B cells and the antibodies they encode is important for understanding the specificity, breadth and duration of immune response to an infection or vaccination. The antibodies isolated could further help design vaccine antigens for raising relevant protective immune responses. However, developing assays to measure and isolate antigen specific memory B cells is technically challenging due to the low frequencies of these cells that exist in the circulating blood. Here we describe a method to develop a direct flow cytometric assay for the detection of Dengue envelope-specific memory B cells using a Dengue envelope specific mouse hybridoma line. We used this flow cytometry assay and a cultured B ELISPOT assay to enumerate Dengue-envelope specific B memory cells from a cohort of Dengue seropositive donors. Furthermore, we were able to culture the single sorted Dengue-envelope specific memory B-cells and differentiate them into plasma cells using cell lines expressing CD40L. The culture supernatants were assayed for antigen binding and the ability of the antibodies to neutralize the cognate dengue virus. Moreover, we successfully isolated the heavy and light Ig sequences and expressed them recombinantly as full length antibodies to reproduce the activity seen in culture supernatants. In conclusion, we have established a robust methodology to enumerate antigen-specific memory B-cells and assay their encoded antibodies for functional characterization.