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In situ humoral selection in human lupus tubulointerstitial inflammation

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In human lupus nephritis, tubulointerstitial inflammation (TII) is associated with in situ expansion of B cells expressing anti-vimentin antibodies (AVAs). The mechanism by which AVAs are selected is unclear. Herein, we demonstrate that AVA somatic hypermutation and selection increase affinity for vimentin. However, enzyme-linked immunosorbent assays (ELISAs) suggested that affinity maturation might be a non-specific consequence of increasing polyreactivity. Subsequent multi-color confocal microscopy studies indicated that while TII AVAs often appeared polyreactive by ELISA, they bound selectively to vimentin fibrils in whole cells or inflamed renal tissue. Using a novel machine learning pipeline (CytoSkaler) to quantify the cellular distribution of antibody staining, we demonstrated that TII AVAs were selected for both enhanced binding and specificity in situ . These data suggest a new approach to assess and define antibody polyreactivity based on quantifying the distribution of binding to native and contextually relevant antigens. ### Competing Interest Statement The authors have declared no competing interest.

Hemagglutinin stalk-reactive antibodies interfere with influenza virus neuraminidase activity by steric hindrance

Journal of Virology, 2019-02-05

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Hemagglutinin (HA) stalk-reactive antibodies are the basis of several current one-shot universal influenza vaccine efforts because they protect against a wide spectrum of influenza virus strains. The appreciated mechanism of protection by HA-stalk antibodies is to inhibit HA stalk reconfiguration, blocking viral fusion and entry. This study shows that HA stalk-reactive antibodies also inhibit neuraminidase (NA) enzymatic activity, prohibiting viral egress. NA inhibition (NI) is evident for an attached substrate but not for unattached small molecule cleavage of sialic acid. This suggests that the antibodies inhibit NA enzymatic activity through steric hindrance, thus limiting NA access to sialic acids when adjacent to HA on whole virions. Consistently, F(ab)2 fragments that occupy reduced area without loss of avidity or disrupted HA/NA interactions show significantly reduced NI activity. Notably, HA stalk binding antibodies lacking NI activity were unable to neutralize viral infection via microneutralization assays. This work suggests that NI activity is an important component of HA-stalk antibody mediated protection.

Identification of antibodies targeting the H3N2 hemagglutinin receptor binding site following vaccination of humans

Cell Reports, 2019-12-24

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Antibodies targeting the receptor binding site (RBS) of the influenza virus hemagglutinin (HA) protein are usually not broadly-reactive because their footprints are typically large and extend to nearby variable HA residues. Here, we identified several human H3N2 HA RBS-targeting monoclonal antibodies (mAbs) that were sensitive to substitutions in conventional antigenic sites and were not broadly-reactive. However, we also identified one H3N2 HA RBS-targeting mAb that was exceptionally broadly reactive despite being sensitive to substitutions in residues outside of the RBS. We determined that similar antibodies are present at measurable levels in the sera of some individuals but that they are inefficiently elicited by conventional vaccines. Our data indicate that some HA RBS-targeting antibodies can be surprisingly effective against variable viral strains even if they are somewhat sensitive to substitutions in HA residues adjacent to the RBS.

An egg-derived sulfated N-Acetyllactosamine glycan is an antigenic decoy of influenza virus vaccines

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Influenza viruses grown in eggs for the purposes of vaccine generation often acquire mutations during egg adaptation or possess differential glycosylation patterns than viruses circulating amongst humans. Here, we report that seasonal influenza virus vaccines possess an egg-derived sulfated N-acetyllactosamine (LacNAc) that is an antigenic decoy. Half of subjects that received an egg-grown vaccine mounted an antibody response against this egg-derived antigen. Egg-binding monoclonal antibodies specifically bind viruses grown in eggs, but not viruses grown in other chicken derived cells, suggesting only egg-grown vaccines can induce anti-LacNAc antibodies. Notably, antibodies against the sulfated LacNAc utilized a restricted antibody repertoire and possessed features of natural antibodies, as most antibodies were IgM and have simple heavy chain complementarity determining region 3. By analyzing a public dataset of influenza virus vaccine induced plasmablasts, we discovered egg-binding public clonotypes that were shared across studies. Together, this study shows that egg-grown vaccines can induce antibodies against an egg-associated glycan, which may divert the host immune response away from protective epitopes.

A public broadly neutralizing antibody class targets a membrane-proximal anchor epitope of influenza virus hemagglutinin

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Broadly neutralizing antibodies against influenza virus hemagglutinin (HA) have the potential to provide universal protection against influenza virus infections. Here, we report a distinct class of broadly neutralizing antibodies targeting an epitope toward the bottom of the HA stalk domain where HA is "anchored" to the viral membrane. Antibodies targeting this membrane-proximal anchor epitope utilized a highly restricted repertoire, which encode for two conserved motifs responsible for HA binding. Anchor targeting B cells were common in the human memory B cell repertoire across subjects, indicating pre-existing immunity against this epitope. Antibodies against the anchor epitope at both the serological and monoclonal antibody levels were potently induced in humans by a chimeric HA vaccine, a potential universal influenza virus vaccine. Altogether, this study reveals an underappreciated class of broadly neutralizing antibodies against H1-expressing viruses that can be robustly recalled by a candidate universal influenza virus vaccine.