Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved variants with substitutions in the spike receptor-binding domain (RBD) that affect its affinity for angiotensin-converting enzyme 2 (ACE2) receptor and recognition by antibodies. These substitutions could also shape future evolution by modulating the effects of mutations at other sites—a phenomenon called epistasis. To investigate this possibility, we performed deep mutational scans to measure the effects on ACE2 binding of all single–amino acid mutations in the Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variant RBDs. Some substitutions, most prominently Asn ⁵⁰¹ →Tyr (N501Y), cause epistatic shifts in the effects of mutations at other sites. These epistatic shifts shape subsequent evolutionary change—for example, enabling many of the antibody-escape substitutions in the Omicron RBD. These epistatic shifts occur despite high conservation of the overall RBD structure. Our data shed light on RBD sequence-function relationships and facilitate interpretation of ongoing SARS-CoV-2 evolution.

Neutralization assays are experimental surrogates for the effectiveness of infection- or vaccine-elicited polyclonal antibodies and therapeutic monoclonal antibodies targeting SARS-CoV-2. However, the measured neutralization can depend on the details of the experimental assay. Here, we systematically assess how ACE2 expression in target cells affects neutralization by antibodies to different spike epitopes in lentivirus pseudovirus neutralization assays. For high ACE2-expressing target cells, receptor-binding domain (RBD) antibodies account for nearly all neutralizing activity in polyclonal human sera. However, for lower ACE2-expressing target cells, antibodies targeting regions outside the RBD make a larger (although still modest) contribution to serum neutralization. These serum-level results are mirrored for monoclonal antibodies: N-terminal domain (NTD) antibodies and RBD antibodies that do not compete for ACE2 binding incompletely neutralize on high ACE2-expressing target cells, but completely neutralize on cells with lower ACE2 expression. Our results show that the ACE2 expression level in the target cells is an important experimental variable, and that high ACE2 expression emphasizes the role of a subset of RBD-directed antibodies.

Conventional influenza virus vaccines induce antibody responses of limited breadth. Whether mRNA-based influenza virus vaccines can induce a superior germinal center (GC) response in humans remains unclear. Here we assessed B cell responses in an observational study of cohorts of healthy young adults receiving a licensed, split-virion or investigative mRNA-based quadrivalent seasonal influenza virus vaccine over two consecutive seasons. mRNA-based vaccines consistently elicited higher antibody titers and frequencies of memory B cells. In the draining lymph nodes, mRNA vaccination stimulated sustained GC reactions that persisted for at least 26 weeks after vaccination in 5 of 13 participants across the two seasons. Proteomic analysis of serum IgG repertoire showed that mRNA vaccination increased the number of vaccine-elicited serum IgG clonotypes and promoted intraclonal expansion within pre-existing clonotypes. B cell lineage analyses further indicated that expanded serum clonotypes map to GC B cell-associated sub-branches, consistent with ongoing GC-driven evolution underlying intraclonal expansion. This repertoire remodeling was accompanied by increased binding breadth against antigenically divergent influenza viruses. These findings reveal a key role for persistent GC responses in broadening the repertoire of vaccine-induced antibodies.

The hemagglutinin (HA) of some influenza viruses can interact with major histocompatibility complex class II (MHC-II), but how these proteins interact is unclear. Here we demonstrate that diverse H5 HAs can use MHC-II to enter cells, with avian MHC-II enabling more efficient entry than human MHC-II for most H5 HAs. To define the molecular interface, we use pseudovirus deep mutational scanning to measure how mutations to H5 HA affect its interaction with tufted duck MHC-II, and identify mutations that restrict HA to exclusively MHC-II or sialic acid receptors. We leverage identification of H5 HA mutations that increase binding to tufted duck MHC-II to determine a 4.8 Å cryo-EM model of the complex. To support the structural model, we measure how all mutations to tufted duck MHC-II affect its interaction with H5 HA, and find the alpha chain is the dominant determinant but beta chain sites near the peptide-binding groove also contribute. To generalize these findings, we use deep mutational scanning to show that a H7 HA interacts with MHC-II similarly to H5 HA. Finally, we show that H1, H2, H3, and H9 HAs interact with avian or human MHC-II, although interactions vary among strains that evolved in different hosts.