Conformational flexibility in neutralization of SARS-CoV-2 by naturally elicited anti-SARS-CoV-2 antibodies

As new variants of SARS-CoV-2 continue to emerge, it is important to assess the cross-neutralizing capabilities of antibodies naturally elicited during wild type SARS-CoV-2 infection. In the present study, we evaluate the activity of nine anti-SARS-CoV-2 monoclonal antibodies (mAbs), previously isolated from convalescent donors infected with the Wuhan-Hu-1 strain, against the SARS-CoV-2 variants of concern (VOC) Alpha, Beta, Gamma, Delta and Omicron. By testing an array of mutated spike receptor binding domain (RBD) proteins, cell-expressed spike proteins from VOCs, and neutralization of SARS-CoV-2 VOCs as pseudoviruses, or as the authentic viruses in culture, we show that mAbs directed against the ACE2 binding site (ACE2bs) are more sensitive to viral evolution compared to anti-RBD non-ACE2bs mAbs, two of which retain their potency against all VOCs tested. At the second part of our study, we reveal the neutralization mechanisms at high molecular resolution of two anti-SARS-CoV-2 neutralizing mAbs by structural characterization. We solve the structures of the Delta-neutralizing ACE2bs mAb TAU-2303 with the SARS-CoV-2 spike trimer and RBD at 4.5 Å and 2.42 Å resolutions, respectively, revealing a similar mode of binding to that between the RBD and ACE2. Furthermore, we provide five additional structures (at resolutions of 4.7 Å, 7.3 Å, 6.4 Å, 3.3 Å, and 6.1 Å) of a second antibody, TAU-2212, complexed with the SARS-CoV-2 spike trimer. TAU-2212 binds an exclusively quaternary epitope, and exhibits a unique, flexible mode of neutralization that involves transitioning between five different conformations, with both arms of the antibody recruited for cross linking intra- and inter-spike RBD subunits. Our study provides additional mechanistic understanding about how antibodies neutralize SARS-CoV-2 and its emerging variants and provides insights on the likelihood of reinfections

Autophagy controls mucus secretion from intestinal goblet cells by alleviating ER stress

Colonic goblet cells are specialized epithelial cells that secrete mucus to physically separate the host and its microbiota, thus preventing bacterial invasion and inflammation. How goblet cells control the amount of mucus they secrete is unclear. We found that constitutive activation of autophagy in mice via Beclin 1 enables the production of a thicker and less penetrable mucus layer by reducing endoplasmic reticulum (ER) stress. Accordingly, genetically inhibiting Beclin 1-induced autophagy impairs mucus secretion, while pharmacologically alleviating ER stress results in excessive mucus production. This ER-stress-mediated regulation of mucus secretion is microbiota dependent and requires the Crohn's-disease-risk gene Nod2. Overproduction of mucus alters the gut microbiome, specifically expanding mucus-utilizing bacteria, such as Akkermansia muciniphila, and protects against chemical and microbial-driven intestinal inflammation. Thus, ER stress is a cell-intrinsic switch that limits mucus secretion, whereas autophagy maintains intestinal homeostasis by relieving ER stress.Appendix on pages e1–e4.</p

Salmonella manipulates the host to drive pathogenicity via induction of interleukin 1β.

Acute gastrointestinal infection with intracellular pathogens like Salmonella Typhimurium triggers the release of the proinflammatory cytokine interleukin 1β (IL-1β). However, the role of IL-1β in intestinal defense against Salmonella remains unclear. Here, we show that IL-1β production is detrimental during Salmonella infection. Mice lacking IL-1β (IL-1β -/-) failed to recruit neutrophils to the gut during infection, which reduced tissue damage and prevented depletion of short-chain fatty acid (SCFA)-producing commensals. Changes in epithelial cell metabolism that typically support pathogen expansion, such as switching energy production from fatty acid oxidation to fermentation, were absent in infected IL-1β -/- mice which inhibited Salmonella expansion. Additionally, we found that IL-1β induces expression of complement anaphylatoxins and suppresses the complement-inactivator carboxypeptidase N (CPN1). Disrupting this process via IL-1β loss prevented mortality in Salmonella-infected IL-1β -/- mice. Finally, we found that IL-1β expression correlates with expression of the complement receptor in patients suffering from sepsis, but not uninfected patients and healthy individuals. Thus, Salmonella exploits IL-1β signaling to outcompete commensal microbes and establish gut colonization. Moreover, our findings identify the intersection of IL-1β signaling and the complement system as key host factors involved in controlling mortality during invasive Salmonellosis

Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19

Sequence variation in PPP1R13L results in a novel form of cardio‐cutaneous syndrome

Dilated cardiomyopathy (DCM) is a life‐threatening disorder whose genetic basis is heterogeneous and mostly unknown. Five Arab Christian infants, aged 4–30 months from four families, were diagnosed with DCM associated with mild skin, teeth, and hair abnormalities. All passed away before age 3. A homozygous sequence variation creating a premature stop codon at PPP1R13L encoding the iASPP protein was identified in three infants and in the mother of the other two. Patients’ fibroblasts and PPP1R13L‐knocked down human fibroblasts presented higher expression levels of pro‐inflammatory cytokine genes in response to lipopolysaccharide, as well as Ppp1r13l‐knocked down murine cardiomyocytes and hearts of Ppp1r13l‐deficient mice. The hypersensitivity to lipopolysaccharide was NF‐κB‐dependent, and its inducible binding activity to promoters of pro‐inflammatory cytokine genes was elevated in patients’ fibroblasts. RNA sequencing of Ppp1r13l‐knocked down murine cardiomyocytes and of hearts derived from different stages of DCM development in Ppp1r13l‐deficient mice revealed the crucial role of iASPP in dampening cardiac inflammatory response. Our results determined PPP1R13L as the gene underlying a novel autosomal‐recessive cardio‐cutaneous syndrome in humans and strongly suggest that the fatal DCM during infancy is a consequence of failure to regulate transcriptional pathways necessary for tuning cardiac threshold response to common inflammatory stressors

Anti-TNFα Treatment Impairs Long-Term Immune Responses to COVID-19 mRNA Vaccine in Patients with Inflammatory Bowel Diseases

Patients with inflammatory bowel disease (IBD) treated with anti-tumor-necrosis factor-alpha (TNFα) exhibited lower serologic responses one-month following the second dose of the COVID-19 BNT162b2 vaccine compared to those not treated with anti-TNFα (non-anti-TNFα) or to healthy controls (HCs). We comprehensively analyzed long-term humoral responses, including anti-spike (S) antibodies, serum inhibition, neutralization, cross-reactivity and circulating B cell six months post BNT162b2, in patients with IBD stratified by therapy compared to HCs. Subjects enrolled in a prospective, controlled, multi-center Israeli study received two BNT162b2 doses. Anti-S levels, functional activity, specific B cells, antigen cross-reactivity, anti-nucleocapsid levels, adverse events and IBD disease score were detected longitudinally. In total, 240 subjects, 151 with IBD (94 not treated with anti-TNFα and 57 treated with anti-TNFα) and 89 HCs participated. Six months after vaccination, patients with IBD treated with anti-TNFα had significantly impaired BNT162b2 responses, specifically, more seronegativity, decreased specific circulating B cells and cross-reactivity compared to patients untreated with anti-TNFα. Importantly, all seronegative subjects were patients with IBD; of those, &gt;90% were treated with anti-TNFα. Finally, IBD activity was unaffected by BNT162b2. Altogether these data support the earlier booster dose administration in these patients

Vancomycin treatment depletes SCFA-producing <i>Clostridia</i> in mice.

16S rRNA sequencing was performed to characterize gut microbiota composition of mice treated with vancomycin for 3 days. Relative abundance (left) and absolute OTU reads (right) of SCFA-producing members of the Clostridia class at the family level. Each symbol represents a mouse. OTU, operational taxonomic unit. These data are representative of 1 experiment. **P t test. Numerical values are in S1 Data. (TIFF)</p

Loss of IL-1β impairs neutrophil recruitment to the gut during <i>Salmonella</i> infection.

(A) Pathway analysis of transcripts that are down-regulated in colonic tissue of Salmonella-infected IL-1β -/- according to GO biological function. Bars represent -log (P value) and dots represent number of genes in pathway. (B) Heatmap depicting differentially expressed innate immune genes with a P C, D) Numbers of NE-positive (C) and myeloperoxidase-positive (D) cells in colonic section of Salmonella-infected mice 4 d.p.i. Each dot represents a mouse. (E) Representative immunohistochemistry images of colonic section from Salmonella-infected mice stained with anti-neutrophil elastase antibody. Scale bar, 50 μm. (F) % of neutrophils out of total circulating WBC and (G) concentration of neutrophils in serum of WT and IL-1β -/- mice infected as indicated 4 d.p.i. *P P P P C and D) Student’s t test. (F and G) One-way ANOVA. d.p.i., days post-infection; S. Tm, Salmonella typhimurium. These data are representative of 2 independent experiments. Numerical values are in S1 Data. The underlying data for this figure can be found at GSE252071. NE, neutrophil elastase; WT, wild-type.</p

Preservation of fatty acid beta-oxidation in <i>IL-1β</i> ^<i>-/-</i> mice inhibits <i>Salmonella</i> growth in vivo.

(A) Pathway analysis of transcripts that are up-regulated in colonic tissue of Salmonella-infected IL-1β -/- according to GO biological function. Bars represent -log (P value) and dots represent number of genes in pathway. (B) Heatmap depicting differentially expressed genes involved in fatty acid oxidation with a P C) Heatmap depicting differentially expressed genes in the glycolysis pathway with a P D) Immunofluorescence microscopy of colonic section from mice. Red staining shows pimonidazole that indicates hypoxia levels. Nuclei were stained with DAPI. Scale bar, 50 μm. (E) Quantification of red signal in (D). Each dot represents a mouse. (F) Salmonella C.F.U. in cecal contents of infected mice 4 d.p.i. treated as indicated. Each dot represents a mouse. **P P E) one-way ANOVA; (F) Mann–Whitney test. d.p.i., days post-infection; S. Tm, Salmonella typhimurium. These data are representative of 2 independent experiments. Numerical values are in S1 Data. The underlying data for this figure can be found at GSE252071.</p

<i>IL-1β</i>^<i>-/-</i> mice co-housed with WT mice are resistant to <i>Salmonella</i> infection.

(A–D) Salmonella C.F.U. 4 days post-infection in cecal content (A), M.L.N. (B), liver (C), and spleen (D) of co-housed mice infected with 107 C.F.U. Salmonella enterica serovar typhimurium (SL1334) 24 h after pretreatment with 20 mg streptomycin. (E) Survival percentage of mice infected orally with Salmonella. (A–D) Each dot represents a mouse. These data are representative of 1 experiment. *P P P A–D) Mann–Whitney test. (E) Mantel–Cox test. C.F.U., colony-forming units; Sep, separately housed; Co, co-housed; d.p.i., days post-infection. Numerical values are in S1 Data. (TIFF)</p