Allelic polymorphism controls autoreactivity and vaccine elicitation of human broadly neutralizing antibodies against influenza virus.

Human broadly neutralizing antibodies (bnAbs) targeting the hemagglutinin stalk of group 1 influenza A viruses (IAVs) are biased for IGHV1-69 alleles that use phenylalanine (F54) but not leucine (L54) within their CDRH2 loops. Despite this, we demonstrated that both alleles encode for human IAV bnAbs that employ structurally convergent modes of contact to the same epitope. To resolve differences in lineage expandability, we compared F54 versus L54 as substrate within humanized mice, where antibodies develop with human-like CDRH3 diversity but are restricted to single VH genes. While both alleles encoded for bnAb precursors, only F54 IGHV1-69 supported elicitation of heterosubtypic serum bnAbs following immunization with a stalk-only nanoparticle vaccine. L54 IGHV1-69 was unproductive, co-encoding for anergic B cells and autoreactive stalk antibodies that were cleared from B cell memory. Moreover, human stalk antibodies also demonstrated L54-dependent autoreactivity. Therefore, IGHV1-69 polymorphism, which is skewed ethnically, gates tolerance and vaccine expandability of influenza bnAbs.

Naive human B cells engage the receptor binding domain of SARS-CoV-2, variants of concern, and related sarbecoviruses.

Initial exposure to a pathogen elicits an adaptive immune response to control and eradicate the threat. Interrogating the abundance and specificity of the naive B cell repertoire drives understanding of how to mount protective responses. Here, we isolated naive B cells from eight seronegative human donors targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD). Single-cell B cell receptor (BCR) sequencing identified diverse gene usage and no restriction on complementarity determining region length. A subset of recombinant antibodies produced by naive B cell precursors bound to SARS-CoV-2 RBD and engaged circulating variants including B.1.1.7, B.1.351, and B.1.617.2, as well as preemergent bat-derived coronaviruses RaTG13, SHC104, and WIV1. By structural characterization of a naive antibody in complex with SARS-CoV-2 spike, we identified a conserved mode of recognition shared with infection-induced antibodies. We found that representative naive antibodies could signal in a B cell activation assay, and by using directed evolution, we could select for a higher-affinity RBD interaction, conferred by a single amino acid change. The minimally mutated, affinity-matured antibodies also potently neutralized SARS-CoV-2. Understanding the SARS-CoV-2 RBD­specific naive repertoire may inform potential responses capable of recognizing future SARS-CoV-2 variants or emerging coronaviruses, enabling the development of pan-coronavirus vaccines aimed at engaging protective germline responses.

Naive human B cells engage the receptor binding domain of SARS-CoV-2, variants of concern, and related sarbecoviruses.

Exposure to a pathogen elicits an adaptive immune response aimed to control and eradicate. Interrogating the abundance and specificity of the naive B cell repertoire contributes to understanding how to potentially elicit protective responses. Here, we isolated naive B cells from 8 seronegative human donors targeting the SARS-CoV-2 receptor-binding domain (RBD). Single B cell analysis showed diverse gene usage with no restricted complementarity determining region lengths. We show that recombinant antibodies engage SARS-CoV-2 RBD, circulating variants, and pre-emergent coronaviruses. Representative antibodies signal in a B cell activation assay and can be affinity matured through directed evolution. Structural analysis of a naive antibody in complex with spike shows a conserved mode of recognition shared with infection-induced antibodies. Lastly, both naive and affinity-matured antibodies can neutralize SARS-CoV-2. Understanding the naive repertoire may inform potential responses recognizing variants or emerging coronaviruses enabling the development of pan-coronavirus vaccines aimed at engaging germline responses.

Spontaneous Glycan Reattachment Following N-Glycanase Treatment of Influenza and HIV Vaccine Antigens.

In cells, asparagine/N-linked glycans are added to glycoproteins cotranslationally, in an attachment process that supports proper folding of the nascent polypeptide. We found that following pruning of N-glycan by the amidase PNGase F, the principal influenza vaccine antigen and major viral spike protein hemagglutinin (HA) spontaneously reattached N-glycan to its de-N-glycosylated positions when the amidase was removed from solution. This reaction, which we term N-glycanation, was confirmed by site-specific analysis of HA glycoforms by mass spectrometry prior to PNGase F exposure, during exposure to PNGase F, and after amidase removal. Iterative rounds of de-N-glycosylation followed by N-glycanation could be repeated at least three times and were observed for other viral glycoproteins/vaccine antigens, including the envelope glycoprotein (Env) from HIV. Covalent N-glycan reattachment was nonenzymatic as it occurred in the presence of metal ions that inhibit PNGase F activity. Rather, N-glycanation relied on a noncovalent assembly between protein and glycan, formed in the presence of the amidase, where linearization of the glycoprotein prevented this retention and subsequent N-glycanation. This reaction suggests that under certain experimental conditions, some glycoproteins can organize self-glycan addition, highlighting a remarkable self-assembly principle that may prove useful for re-engineering therapeutic glycoproteins such as influenza HA or HIV Env, where glycan sequence and structure can markedly affect bioactivity and vaccine efficacy.