SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

Abstract: The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era

Targeting Tumor Necrosis Factor-α with Adalimumab: Effects on Endothelial Activation and Monocyte Adhesion

<div><p>Objective</p><p>It is well known that atherosclerotic inflammatory vascular disease is critically driven by oxidized lipids and cytokines. In this regard, tumor necrosis factor (TNF)-α is known as a crucial mediator of early pro-atherosclerotic events. Epidemiologic data suggest that blockade of TNF-α has beneficial effects on vascular outcomes in patients with rheumatoid arthritis, however, detailed mechanistic studies are still lacking. This study aims to elucidate effects of TNF-α blockade by adalimumab–which is approved for several inflammatory disorders–on endothelial activation and monocyte adhesion under pro-atherosclerotic conditions.</p><p>Methods and Results</p><p>Phorbol myristate acetate (PMA) differentiated THP-1 macrophages were stimulated with oxidized low density lipoprotein and subsequent analysis of this conditioned media (oxLDL CM) revealed a strong release of TNF-α. The TNF-α rich supernatant led to activation of human umbilical vein endothelial cells (HUVEC) as shown by enhanced expression of major adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin which was suppressed by the TNF-α inhibitor adalimumab. Accordingly, adalimumab effectively prevented THP-1 monocyte adhesion to endothelial cells under static as well as under flow conditions. Furthermore, adalimumab suppressed endothelial leakage as shown by Evan's blue diffusion across a confluent endothelial monolayer. Of note, after intraperitoneal injection we detected abundant deposition of fluorophore-labelled adalimumab in atherosclerotic plaques of hypercholesterolemic mice.</p><p>Conclusion</p><p>Our results show that adalimumab prevents major inflammatory effects of TNF-α on endothelial activation, endothelial monocyte adhesion, endothelial leakage and therefore extends the therapeutic options of adalimumab to limit vascular inflammation.</p></div

The lipopeptide MALP-2 promotes collateral growth

Beyond their role in pathogen recognition and the initiation of immune defense, Toll-like receptors (TLRs) are known to be involved in various vascular processes in health and disease. We investigated the potential of the lipopeptide and TLR2/6 ligand macrophage activating protein of 2-kDA (MALP-2) to promote blood flow recovery in mice. Hypercholesterolemic apolipoprotein E (Apoe)-deficient mice were subjected to microsurgical ligation of the femoral artery. MALP-2 significantly improved blood flow recovery at early time points (three and seven days), as assessed by repeated laser speckle imaging, and increased the growth of pre-existing collateral arteries in the upper hind limb, along with intimal endothelial cell proliferation in the collateral wall and pericollateral macrophage accumulation. In addition, MALP-2 increased capillary density in the lower hind limb. MALP-2 enhanced endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) release from endothelial cells and improved the experimental vasorelaxation of mesenteric arteries ex vivo. In vitro, MALP-2 led to the up-regulated expression of major endothelial adhesion molecules as well as their leukocyte integrin receptors and consequently enhanced the endothelial adhesion of leukocytes. Using the experimental approach of femoral artery ligation (FAL), we achieved promising results with MALP-2 to promote peripheral blood flow recovery by collateral artery growth

OxLDL-dependent release of TNF-α and other inflammatory factors from THP-1 macrophages.

<p>TNF-α release from (A) THP-1 macrophages following 48 hours or (B) primary human macrophages following 24 hours of oxLDL (25 and 50 μg/mL) stimulation was determined by ELISA. LPS (100 ng/mL) was used as positive control. *P<0.05, **P<0.01, ***P<0.001 vs. control, n = 4–6 replicated experiments. (C) Conditioned medium from THP-1 macrophages following 48 hours of oxLDL (25 μg/mL) was analyzed by protein array. Conditioned medium from unstimulated THP-1 macrophages were used as control. Bar graph showing densitometric analysis of array membranes specific for inflammation-related factors. Factors with more than 2-fold increase after stimulation are indicated in the graph.</p

Effect of TNF-α inhibition with adalimumab on endothelial permeability.

<p>A confluent monolayer of HUVECs on the membrane of transwell inserts (pore size 0.4 μm) was incubation with (A) conditioned media from oxLDL-stimulated THP-1 macrophages (oxLDL CM) with or without adalimumab (ada) or (B) with TNF-α (10 ng/mL) for 4 hours. Diffusion of Evan´s blue bound BSA across the endothelial monolayer from the luminal to the abluminal chamber was quantified by measuring absorbance at 620 nm of the abluminal chamber after additional 3 hours. (C) VE-cadherin immunofluorescence staining visualizes endothelial cell-to-cell adherens junctions. Conditioned medium from unstimulated THP-1 macrophages (control CM), IgG isotype (1 μg/mL) and medium were used as control. Scale bar = 25 μm. Representative pictures are shown. *P<0.05 vs. control CM or medium, ^#P<0.05 vs. oxLDL CM, n = 3–4 replicated experiments.</p

Effect of TNF-α inhibition with adalimumab on adhesion of THP-1 monocytes to endothelial cells.

<p>Adhesion assay under static conditions. (A) Fluorescence images depicting CellTracker green-labelled THP-1 monocytes on a HUVECs monolayer after incubation with conditioned media from oxLDL-stimulated THP-1 macrophages (oxLDL CM) with or without adalimumab (ada) for 4 hours followed by the addition of CellTracker green-labelled THP-1 monocytes. Pictures before and after washing with basal medium are shown. (B) Adherent cells per high power field were quantified after washing. (C) HUVECs monolayer was stimulated with TNF-α (10 ng/mL) for 4 hours followed by the addition of CellTracker green-labelled THP-1 monocytes. Adherent cells per high power field were quantified after washing. Adhesion assay under flow conditions. (D) Phase contrast images of THP-1 monocytes on a HUVECs monolayer after incubation with conditioned media from oxLDL-stimulated THP-1 macrophages (oxLDL CM) with or without adalimumab (ada) for 4 hours followed by the addition of THP-1 monocytes with a flow rate of 0.53 mL/min (0.5 dyne/cm²). (E) Adherent cells per high power field were quantified after washing. (F) HUVECs monolayer was stimulated with TNF-α (10 ng/mL) for 4 hours followed by the addition of THP-1 monocytes with a flow rate of 0.53 mL/min (0.5 dyne/cm²). Adherent cells per high power field were quantified after washing. Conditioned medium from unstimulated THP-1 macrophages (control CM), IgG isotype (1 μg/mL) and medium were used as control. Scale bar = 200 μm. Representative pictures are shown. ***P<0.001 vs. control CM or medium, ^##P<0.01, ^###P<0.001 vs. oxLDL CM, n = 3–4 replicated experiments.</p

The lipopeptide MALP-2 promotes collateral growth

Beyond their role in pathogen recognition and the initiation of immune defense, Toll-like receptors (TLRs) are known to be involved in various vascular processes in health and disease. We investigated the potential of the lipopeptide and TLR2/6 ligand macrophage activating protein of 2-kDA (MALP-2) to promote blood flow recovery in mice. Hypercholesterolemic apolipoprotein E (Apoe)-deficient mice were subjected to microsurgical ligation of the femoral artery. MALP-2 significantly improved blood flow recovery at early time points (three and seven days), as assessed by repeated laser speckle imaging, and increased the growth of pre-existing collateral arteries in the upper hind limb, along with intimal endothelial cell proliferation in the collateral wall and pericollateral macrophage accumulation. In addition, MALP-2 increased capillary density in the lower hind limb. MALP-2 enhanced endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) release from endothelial cells and improved the experimental vasorelaxation of mesenteric arteries ex vivo. In vitro, MALP-2 led to the up-regulated expression of major endothelial adhesion molecules as well as their leukocyte integrin receptors and consequently enhanced the endothelial adhesion of leukocytes. Using the experimental approach of femoral artery ligation (FAL), we achieved promising results with MALP-2 to promote peripheral blood flow recovery by collateral artery growth

Monocyte subpopulation profiling indicates CDK6-derived cell differentiation and identifies subpopulation-specific miRNA expression sets in acute and stable coronary artery disease

Coronary artery disease (CAD) is a long-lasting inflammatory disease characterized by monocyte migration into the vessel wall leading to clinical events like myocardial infarction (MI). However, the role of monocyte subsets, especially their miRNA-driven differentiation in this scenario is still in its infancy. Here, we characterized monocyte subsets in controls and disease phenotypes of CAD and MI patients using flow cytometry and miRNA and mRNA expression profiling using RNA sequencing. We observed major differences in the miRNA profiles between the classical (CD14++CD16-) and nonclassical (CD14+CD16++) monocyte subsets irrespective of the disease phenotype suggesting the Cyclin-dependent Kinase 6 (CDK6) to be an important player in monocyte maturation. Between control and MI patients, we found a set of miRNAs to be differentially expressed in the nonclassical monocytes and targeting CCND2 (Cyclin D2) that is able to enhance myocardial repair. Interestingly, miRNAs as miR-125b playing a role in vascular calcification were differentially expressed in the classical subset in patients suffering from CAD and not MI in comparison to control samples. In conclusion, our study describes specific peculiarities of monocyte subset miRNA expression in control and diseased samples and provides basis to further functional analysis and to identify new cardiovascular disease treatment targets.</p