Efficient Isolation of Monoclonal Antibodies from Antigen-specific Human Plasmablasts and Memory B Cells from a Pneumococcal Conjugate Vaccinee

Abstract

Abstract Community-acquired pneumonia is a leading infectious cause of hospitalization, the annual incidence of Pneumococcal pneumonia is 24.8 cases per 10,000 adults in USA. Pneumococcal conjugate vaccine (PCV13) is currently used in clinic to prevent pneumococcal disease. We isolated plasmablast cells from a Pneumococcal conjugate vaccinee 7 days post vaccination to understand humoral responses. The plasmablast cells were single-sorted into 96 well PCR plates and applied to direct molecular cloning for PCR amplification of antibody sequences. Paired antibody heavy and light chain variable region sequences were achieved from 35 out of 48 wells with molecular cloning success rate of 72.9%. The antibody sequences were recombinantly expressed as human IgG1, and 17 out of 35 hits were positive for vaccine antigen by ELSIA s giving a positive rate of 48.9%. We also isolated memory B cells from the same donor 28 days post vaccination. The memory B cells were single-sorted into 96 well cell culture plates pre-seeded with CD40L-expressing feeder cells in complete RPMI 1640 medium complemented with IL21 and cultured for two weeks. The culture supernatants were tested in ELISA and 7 wells were identified as antigen-specific hits. Paired heavy and light chain antibody sequences were amplified from all of the 7 hits, all of them were successfully cloned and recombinantly produced as human IgG1 and tested in ELISA. 6 out of the 7 hits (85.7%) turned out to be vaccine antigen specific. We will further compare and discuss the clonal specificities isolated from plasmablast and memory B cells to understand the clone’s participation in immune responses to vaccination.