Multi-step antibody class switching in a primary human response is restricted afterIGHG2and dependent on B cell maturation stage

Class switch recombination (CSR) allows the formation of functionally specialized antibodies. Understanding of CSR dynamics is key for better design and prediction of vaccines to protect mucosal surfaces. To investigate CSR in a primary human immune response under a controlled setting, we sampled healthy volunteers without COVID-19 history every other day during the first three weeks after SARS-CoV-2 vaccination, with additional time points up to six months. Leveraging bulk and single-cell B cell receptor repertoires, single-cell transcriptomics, and immunophenotyping, we uncover paradigm-shifting insights into CSR. Newly activated B cells produce sterile transcripts of all antibody isotype constant regions (IGHC) simultaneously up toIGHG2, challenging the view that sterile transcription occurs for only a singleIGHCgene at a time. CSR follows a multistep progression along theIGHClocus; in this challenge vaccine-induced B cells switch fromIGHMtoIGHG3andIGHG1, followed by a subsequent switching toIGHA1andIGHG2after secondary immunization.IGHA2clones require pre-switching toIGHA1clones. Notably, switching tendency, measured byIGHCsterile transcription, is memory B cell subtype dependent, particularly beyondIGHG2. Contrary to other vaccines, antigen-specific B cells are enriched in DN2, Cmem2 and DN4 subtypes after immunization. We also observe a temporal decoupling of CSR and somatic hypermutation (SHM), with the latter detectable only after six months post-immunization. Our data describes the dynamics between CSR, SHM, sterile transcription and B cell memory development during a human primary response, challenges textbook models of CSR and offers new insights to aid control of CSR direction.