All animals and plants dynamically attach and remove O-linked beta-N-acetylglucosamine (O-GlcNAc) at serine and threonine residues on myriad nuclear and cytoplasmic proteins. O-GlcNAc cycling, which is tightly regulated by the concerted actions of two highly conserved enzymes, serves as a nutrient and stress sensor. On some proteins, O-GlcNAc competes directly with phosphate for serine/threonine residues. Glycosylation with O-GlcNAc modulates signalling, and influences protein expression, degradation and trafficking. Emerging data indicate that O-GlcNAc glycosylation has a role in the aetiology of diabetes and neurodegeneration.

An ideal anti-SARS-CoV-2 antibody would resist viral escape [1][2][3] , have activity against diverse SARS-related coronaviruses (sarbecoviruses) [4][5][6][7] , and be highly protective through viral neutralization [8][9][10][11] and effector functions 12,13 . Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a tradeoff between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a previously undescribed cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we characterize one potent RBM antibody (S2E12 8 ) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth, and potency among antibodies targeting the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.The most potently neutralizing antibodies to SARS-CoV-2-including those in clinical use 14 and dominant in polyclonal sera 15,16 -target the spike receptor-binding domain (RBD). Mutations in the RBD that reduce binding by antibodies have emerged among SARS-CoV-2 variants [17][18][19][20][21] , highlighting the need for antibodies and vaccines that are robust to viral escape. We have previously described an antibody, S309 4 , that exhibits potent effector functions and neutralizes all current SARS-CoV-2 variants 22,23 and the divergent sarbecovirus SARS-CoV-1. S309 forms the basis for an antibody therapy (VIR-7831, recently renamed sotrovimab) that has received Emergency Use Authorization from the FDA for treatment of COVID-19 24 . Longer term, antibodies with broad activity across SARS-related coronaviruses (sarbecoviruses) would be useful to combat potential future spillovers 6 . These efforts would be aided by a systematic understanding of the relationships among antibody epitope,

The spillovers of β-coronaviruses in humans and the emergence of SARS-CoV-2 variants highlight the need for broad coronavirus countermeasures. We describe five monoclonal antibodies (mAbs) cross-reacting with the stem helix of multiple β-coronavirus spike glycoproteins isolated from COVID-19 convalescent individuals. Using structural and functional studies we show that the mAb with the greatest breadth (S2P6) neutralizes pseudotyped viruses from three different subgenera through inhibition of membrane fusion and delineate the molecular basis for its cross-reactivity. S2P6 reduces viral burden in hamsters challenged with SARS-CoV-2 through viral neutralization and Fc-mediated effector functions. Stem helix antibodies are rare, oftentimes of narrow specificity and can acquire neutralization breadth through somatic mutations. These data provide a framework for structure-guided design of pan-β-coronavirus vaccines eliciting broad protection.

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Citrobacter rodentium, a murine model pathogen for human enteropathogenic Escherichia coli, predominantly colonizes the lumen and mucosal surface of the colon and cecum and causes crypt hyperplasia and mucosal inflammation. Mice infected with C. rodentium develop a secretory immunoglobulin A (IgA) response, but the role of B cells or secretory antibodies in host defense is unknown. To address this question, we conducted oral C. rodentium infections in mice lacking B cells, IgA, secreted IgM, polymeric Ig receptor (pIgR), or J chain. Normal mice showed peak bacterial numbers in colon and feces at 1 week and bacterial eradication after 3 to 4 weeks. B-cell-deficient mice were equally susceptible initially but could not control infection subsequently. Tissue responses showed marked differences, as infection of normal mice was accompanied by transient crypt hyperplasia and mucosal inflammation in the colon and cecum at 2 but not 6 weeks, whereas B-cell-deficient mice had few mucosal changes at 2 weeks but severe epithelial hyperplasia with ulcerations and mucosal inflammation at 6 weeks. The functions of B cells were not mediated by secretory antibodies, since mice lacking IgA or secreted IgM or proteins required for their transport into the lumen, pIgR or J chain, cleared C. rodentium normally. Nonetheless, systemic administration of immune sera reduced bacterial numbers significantly in normal and pIgR-deficient mice, and depletion of IgG abrogated this effect. These results indicate that host defense against C. rodentium depends on B cells and IgG antibodies but does not require production or transepithelial transport of IgA or secreted IgM.

The transcriptional co-activator PGC-1␣ plays a key role in regulating gene expression required for the stress response in neurons, adaptive thermogenesis in brown adipose tissue, muscle fiber-type switching, and multiple metabolic pathways in the liver (1, 2). PGC-1␣ responds to oxidative stress and activates expression of reactive oxygen species (ROS) 2 detoxifying enzymes. In fact, PGC-1␣ knock-out mice are more sensitive to oxidative stress, particularly in neuronal tissues (3). PGC-1␣ regulates mitochondrial biosynthesis and respiration by stimulating the activity of a number of transcription factors including NRF-1/2, the peroxisome proliferator-activated receptors, the retinoid X receptors, and estrogen-related receptor ␣ (1). In the liver, PGC-1␣ acts as a nutrient sensor through deacetylation by SIRT1 in response to fasting or pyruvate. Deacetylation of PGC-1␣ alters HNF4␣-dependent expression of the gluconeogenicenzymesPepckandG6pc(4).PGC-1␣alsoenhancesFoxO1-dependent activation of gluconeogenesis (5).O-GlcNAc is a post-translational addition of ␤-N-acetylglucosamine to serine and threonine residues of nuclear and cytoplasmic proteins. In addition to its cellular location, it is unlike classical glycosylation in that it is not elongated and cycles on and off proteins dynamically. In fact, O-GlcNAc is functionally more analogous to phosphorylation and is crucial to cell signaling processes including insulin signaling, cell cycle progression, transcription and translation, protein turnover, and stress responses (6).Insulin resistance and altered glucose metabolism damage cells of the nervous system, heart, vasculature, and kidneys (7). Insulin resistance can be caused by elevated flux through the UDP-GlcNAc synthesis pathway, which provides the donor sugar nucleotide for GlcNAcylation (8). In cultured adipocytes, elevating O-GlcNAc both by inhibition of O-GlcNAcase and by increasing UDP-GlcNAc levels decreased insulin-mediated glucose uptake (9). One mechanism of this phenomenon appears to involve a novel phosphatidylinositol 3,4,5-trisphosphate (PIP 3 )-binding domain of OGT. Insulin stimulation recruits OGT to the plasma membrane through interaction with PIP 3 , causing GlcNAcylation of the insulin receptor and insulin receptor substrate (10). In mice, transgenic overexpression of OGT in fat or muscle caused insulin resistance and hyperleptinemia (11). In Caenorhabditis elegans, knocking down OGT alters nutrient storage and dauer formation in a daf-2 (insulin receptor homolog) temperature-sensitive mutant.In addition to peripheral insulin resistance, OGT also mediates the paradoxical activation of hepatic gluconeogenesis. Hyperglycemia results in increased UDP-GlcNAc levels in the liver, increasing GlcNAcylation of FoxO (12) and also increasing the GlcNAcylation of CREB-regulated transcription co-activator 2 (CRTC2 or TORC2, transducer of regulated cylic adenosine monophosphate response element-binding protein 2) (13). The transcription factor FoxO, which regulates meta-

The protozoan pathogen Giardia is an important cause of parasitic diarrheal disease worldwide. It colonizes the lumen of the small intestine, suggesting that effective host defenses must act luminally. Immunoglobulin A (IgA) antibodies are presumed to be important for controlling Giardia infection, but direct evidence for this function is lacking. B-cell-independent effector mechanisms also exist and may be equally important for antigiardial host defense. To determine the importance of the immunoglobulin isotypes that are transported into the intestinal lumen, IgA and IgM, for antigiardial host defense, we infected gene-targeted mice lacking IgA-expressing B-cells, IgM-secreting B-cells, or all B-cells as controls with Giardia muris or Giardia lamblia GS/M-83-H7. We found that IgA-deficient mice could not eradicate either G. muris or G. lamblia infection, demonstrating that IgA is required for their clearance. Furthermore, although neither B-cell-deficient nor IgA-deficient mice could clear G. muris infections, IgA-deficient mice controlled infection significantly better than B-cell-deficient mice, suggesting the existence of B-cell-dependent but IgA-independent antigiardial defenses. In contrast, mice deficient for secreted IgM antibodies cleared G. muris infection normally, indicating that they have no unique functions in antigiardial host defense. These data, together with the finding that B-cell-deficient mice have some, albeit limited, residual capacity to control G. muris infection, show that IgA-dependent host defenses are central for eradicating Giardia spp. Moreover, B-cell-dependent but IgAindependent and B-cell-independent antigiardial host defenses exist but are less important for controlling infection.