Analysis of TLR7/8 Fentanyl Vaccination on the Humoral Compartment by Single Cell Sequencing of the B Cell Receptor

Fentanyl overdose deaths are on the rise from intentional use or accidental exposure. In this context, additional treatment options to Narcan are being explored. One FDA approved alternative is the development of monoclonal antibodies; for fentanyl treatment or as a therapeutic. Our project aimed to use our toll-like receptor 7/8 (TLR 7/8) adjuvanted Fentanyl vaccine to generate monoclonal antibodies against fentanyl. We anticipated the vaccine would change the antibodies produced when compared to naive conditions and this would suggest potential candidates for future antibody treatment. To accomplish this, mice were vaccinated with a TLR 7/8 fentanyl hapten vaccine, and lymph nodes were harvested and processed into a single cell suspension. Using antigen specific magnetic enrichment and single cell sorting, we isolated fentanyl specific B cells. From these cells, we amplified cDNA for each individual cell followed by two rounds of nested PCR using primers specific for heavy and light chain regions of the B cell receptor (BCR). Positive samples were identified by gel electrophoresis followed by Sanger sequencing and BCR allele annotation. Using bioinformatic analysis, we were able to explore allele usage, isotype/class switching, somatic hypermutation rate, and CDR3 amino acids. The results showed heightened allele selection, greater class switching to IgG, elevated somatic hypermutation, and conserved CDR3 regions after vaccination indicative of an enhanced germinal center response. Meta analysis of the increased antibody modifications indicates significant differences which allow us to identify possible monoclonal antibodies for expression and subsequent efficacy studies.