Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha’s small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic.

Transient central retina artery occlusion in patients undergone intravitreal anti-vascular endothelial growth factor injections

Purpose To evaluate the occurrence of transient central retinal artery occlusion following intravitreal anti-vascular endothelial growth factor injection. Methods Prospective, observational study of 807 patients (807 eyes) who were given intravitreal injections of ranibizumab or aflibercept to treat any cause of retinal vascular diseases between 1 January 2017 and 30 November 2018 at the Federal Fluminense University Hospital in Niteroi, and a private facility in Rio de Janeiro, Brazil. Patients who did not present transient central retinal artery occlusion were excluded. Results Among 4069 injections, only 18 patients (0.44%) presented transient central retinal artery occlusion, 14 mild cases (77.7%), and 4 severe cases (22.3%). The clinical factors associated with more severe cases of transient central retinal artery occlusion were the duration of the transient central retinal artery occlusion ( p = 0.001), number of prior injections ( p = 0.01), and a positive carotid Doppler test ( p = 0.01). Twelve cases (66.6%) had positive carotid artery obstruction (atheroma plaque size ≥70%) while 6 cases (33.3%) had negative carotid artery obstruction (atheroma plaque size <70%). The age group >60 years old ( p = 0.06), cup/disc ratio >0.6 ( p = 0.06), and pseudophakic lens status were also factors with association with transient central retinal artery occlusion, although did not meet criteria for statistical significance. The only patient who experienced a recurrent episode of transient central retinal artery occlusion had diabetic macular edema, positive carotid Doppler test, and cup/optic disc ratio >0.6. Conclusion Transient central retinal artery occlusion is a rare adverse event that can appear in patients with retinal vascular disease receiving anti-vascular endothelial growth factor therapy. The atheroma plaque size and the number of prior injections can be associated with the severity of the event.

Anti-Galectin-2 antibody treatment reduces atherosclerotic plaque size and alters macrophage polarity

Galectins have numerous cellular functions in immunity and inflammation. Short-term galectin-2 blockade in ischaemia-induced arteriogenesis shifts macrophages to an anti-inflammatory phenotype and improves perfusion. Galectin-2 may also affect other macrophage related cardiovascular diseases. This study aims to elucidate the effects of Galectin-2 inhibition in atherosclerosis. ApoE -/- mice were given a high cholesterol diet (HCD) for 12 weeks. After six weeks of HCD, intermediate atherosclerotic plaques were present. To study the effects of anti-Gal2 nanobody treatment on the progression of existing atherosclerosis, treatment with two llama derived anti-Gal2 nanobodies (clones- 2H8 and 2C10), or vehicle was given for the remaining 6 weeks. Galectin-2 inhibition reduced the progression of existing atherosclerosis. Atherosclerotic plaque area in the aortic root was decreased, especially so in mice treated with 2C10 nanobodies. This clone reduced atherosclerosis severity as reflected by a decrease in fibrous cap atheromas in addition to decreases in plaque size. The number of plaque resident macrophages was unchanged, however, there was a significant increase in the fraction of CD206+ macrophages. 2C10 treatment also increased plaque α-smooth muscle content, and Gal-2 may have a role in modulating the inflammatory status of smooth muscle cells. Remarkably, both treatments reduced serum cholesterol concentrations including reductions in VLDL, LDL, and HDL whilst triglyceride concentrations were unchanged. Prolonged treatment with anti-Gal-2 nanobodies reduced plaque size, slowed plaque progression, and modified the phenotype of plaque macrophages toward an anti-inflammatory profile. These results hold promise for future macrophage modulating therapeutic interventions that promote arteriogenesis and reduce atherosclerosis.

Studies on Growth Characteristics and Cross-Neutralization of Wild-Type and Delta SARS-CoV-2 From Hisar (India)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly evolved to generate several antigenic variants. These variants have raised concerns whether pre-existing immunity to vaccination or prior infection would be able to protect against the newly emerging SARS-CoV-2 variants or not. We isolated SARS-CoV-2 from the coronavirus disease 2019 (COVID-19)-confirmed patients in the beginning of the first (April/May 2020) and second (April/May 2021) waves of COVID-19 in India (Hisar, Haryana). Upon complete nucleotide sequencing, the viruses were found to be genetically related with wild-type (WT) and Delta variants of SARS-CoV-2, respectively. The Delta variant of SARS-CoV-2 produced a rapid cytopathic effect (24–36 h as compared to 48–72 h in WT) and had bigger plaque size but a shorter life cycle (~6 h as compared to the ~8 h in WT). Furthermore, the Delta variant achieved peak viral titers within 24 h as compared to the 48 h in WT. These evidence suggested that the Delta variant replicates significantly faster than the WT SARS-CoV-2. The virus neutralization experiments indicated that antibodies elicited by vaccination are more efficacious in neutralizing the WT virus but significantly less potent against the Delta variant. Our findings have implications in devising suitable vaccination, diagnostic and therapeutic strategies, besides providing insights into understanding virus replication and transmission.

Comparison of Plaque Size, Thermal Stability, and Replication Rate among SARS-CoV-2 Variants of Concern

SARS-CoV-2, like other RNA viruses, has a propensity for genetic evolution owing to the low fidelity of its viral polymerase. This evolution results in the emergence of novel variants with different characteristics than their ancestral strain. Several recent reports have described a series of novel SARS-CoV-2 variants. Some of these have been identified as variants of concern (VOCs), including alpha (B.1.1.7, Clade GRY), beta (B.1.351, Clade GH), gamma (P.1, Clade GR), and delta (B.1.617.2, Clade G). VOCs are likely to have some effect on transmissibility, antibody evasion, and changes in therapeutic or vaccine effectiveness. However, the physiological and virological understanding of these variants remains poor. We demonstrated that these four VOCs exhibited differences in plaque size, thermal stability at physiological temperature, and replication rates. The mean plaque size of beta was the largest, followed by those of gamma, delta, and alpha. Thermal stability, evaluated by measuring infectivity and half-life after prolonged incubation at physiological temperature, was correlated with plaque size in all variants except alpha. However, despite its relatively high thermal stability, alpha's small plaque size resulted in lower replication rates and fewer progeny viruses. Our findings may inform further virological studies of SARS-CoV-2 variant characteristics, VOCs, and variants of interest. These studies are important for the effective management of the COVID-19 pandemic.

Deficiency of myeloid prolyl hydroxylase domain proteins aggravates atherogenesis via macrophage apoptosis and paracrine fibrotic signaling

Background Atherosclerotic plaque hypoxia is detrimental for macrophage function. Prolyl hydroxylases (PHDs) initiate cellular hypoxic responses, possibly influencing macrophage function in plaque hypoxia. Thus, we aimed to elucidate the role of myeloid PHDs in atherosclerosis. Methods Myeloid specific PHD knockout (PHDko) mice were fed high cholesterol diet for 6–12 weeks to induce atherosclerosis. Plaque parameters, e.g. plaque size and macrophage content, were analyzed. Bulk and single cell RNA sequencing was performed on PHD2 BMDMs and plaque macrophages, respectively. Results Aortic root plaque size was augmented 2.6fold in PHD2cko, and 1.4-fold in PHD3ko, but not in PHD1ko mice compared to controls. Macrophage apoptosis was promoted in PHD2cko and PHD3ko mice in vitro and in vivo, via the HIF1α/BNIP3 axis. Bulk and single cell RNA data of PHD2cko bone-marrow-derived macrophages (BMDM) and plaque macrophages, respectively, confirmed these findings and were validated by siRNA silencing. Human plaque BNIP3 mRNA associated with plaque necrotic core, suggesting similar adverse effects. Further, PHD2cko plaques displayed enhanced fibrosis, independent of macrophage MMP activity, collagen secretion or proliferation and of SMC collagen production, or proliferation. Rather, PHD2cko BMDMs enhanced fibroblast collagen secretion in a paracrine manner. Nichenet in silico analysis of macrophage-fibroblast communication predicted SPP1 signaling as regulator, in line with enhanced plaque SPP1 protein content, and SPP1 mRNA in TREM2-foamy plaque macrophages, but not in neutrophils. Conclusion Myeloid PHD2cko and PHD3ko enhanced plaque growth, macrophage apoptosis, and PHD2cko activated paracrine collagen secretion by fibroblasts. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): NWO, Leducq

Shigella flexneri Diguanylate Cyclases Regulate Virulence

Shigella flexneri is an intracellular human pathogen that invades colonic cells and causes bloody diarrhea. S. flexneri evolved from commensal Escherichia coli , and genome comparisons reveal that S. flexneri has lost approximately 20% of its genes through the process of pathoadaptation, including a disproportionate number of genes associated with the turnover of the nucleotide-based second messenger cyclic di-guanosine monophosphate (c-di-GMP); however, the remaining c-di-GMP turnover enzymes are highly conserved. C-di-GMP regulates many behavioral changes in other bacteria in response to changing environmental conditions, including biofilm formation, but this signaling system has not been examined in S. flexneri . In this study, we expressed VCA0956, a constitutively active c-di-GMP synthesizing diguanylate cyclase (DGC) from Vibrio cholerae , in S. flexneri to determine if virulence phenotypes were regulated by c-di-GMP. We found that expressing VCA0956 in S. flexneri increased c-di-GMP levels, and this corresponds with increased biofilm formation, and reduced acid resistance, host cell invasion, and plaque size. We examined the impact of VCA0956 expression on the S. flexneri transcriptome, and found that genes related to acid resistance were repressed, and this corresponded with decreased survival to acid shock. We also found that individual S. flexneri DGC mutants exhibit reduced biofilm formation, reduced host cell invasion and plaque size, as well as increased resistance to acid shock. This study highlights the importance of c-di-GMP signaling in regulating S. flexneri virulence phenotypes Importance The intracellular human pathogen Shigella causes dysentery, resulting in as many as one million deaths per year. Currently, there is no approved vaccine for the prevention of shigellosis, and the incidence of antimicrobial resistance amongst Shigella species is on the rise. Here, we explore how the widely conserved c-di-GMP bacterial signaling system alters Shigella behaviors associated with pathogenesis. We find that expressing or removing enzymes associated with c-di-GMP synthesis results in changes in Shigella ’s ability to form biofilms, invade host cells, form lesions in host cell monolayers, and resist acid stress.

Whole-Body Prolyl Hydroxylase Domain (PHD) 3 Deficiency Increased Plasma Lipids and Hematocrit Without Impacting Plaque Size in Low-Density Lipoprotein Receptor Knockout Mice

Background and aims: Atherosclerosis is an important cause of clinical cardiovascular events. Atherosclerotic plaques are hypoxic, and reoxygenation improves plaque phenotype. Central players in hypoxia are hypoxia inducible factors (HIF) and their regulators, HIF-prolyl hydroxylase (PHD) isoforms 1, 2, and 3. PHD inhibitors, targeting all three isoforms, are used to alleviate anemia in chronic kidney disease. Likewise, whole-body PHD1 and PHD2ko ameliorate hypercholesterolemia and atherogenesis. As the effect of whole-body PHD3 is unknown, we investigated the effects of germline whole-body PHD3ko on atherosclerosis.Approach and Results: To initiate hypercholesterolemia and atherosclerosis low-density lipoprotein receptor knockout (LDLrko) and PHD3/LDLr double knockout (PHD3dko), mice were fed a high-cholesterol diet. Atherosclerosis and hypoxia marker pimonidazole were analyzed in aortic roots and brachiocephalic arteries. In contrast to earlier reports on PHD1- and PHD2-deficient mice, a small elevation in the body weight and an increase in the plasma cholesterol and triglyceride levels were observed after 10 weeks of diet. Dyslipidemia might be explained by an increase in hepatic mRNA expression of Cyp7a1 and fatty acid synthase, while lipid efflux of PHD3dko macrophages was comparable to controls. Despite dyslipidemia, plaque size, hypoxia, and phenotype were not altered in the aortic root or in the brachiocephalic artery of PHD3dko mice. Additionally, PHD3dko mice showed enhanced blood hematocrit levels, but no changes in circulating, splenic or lymphoid immune cell subsets.Conclusion: Here, we report that whole-body PHD3dko instigated an unfavorable lipid profile and increased hematocrit, in contrast to other PHD isoforms, yet without altering atherosclerotic plaque development.

DEFICIENCY OF MYELOID PHD PROTEINS AGGRAVATES ATHEROGENESIS VIA MACROPHAGE APOPTOSIS AND PARACRINE FIBROTIC SIGNALING: Atherogenic effects of myeloid PHD knockdown

Aims Atherosclerotic plaque hypoxia is detrimental for macrophage function. Prolyl hydroxylases (PHDs) initiate cellular hypoxic responses, possibly influencing macrophage function in plaque hypoxia. Thus, we aimed to elucidate the role of myeloid PHDs in atherosclerosis. Methods & Results Myeloid specific PHD knockout (PHDko) mice were obtained via bone marrow transplantation (PHD1ko, PHD3ko) or conditional knockdown through lysozyme M-driven Cre recombinase (PHD2cko). Mice were fed high cholesterol diet for 6-12 weeks to induce atherosclerosis. Aortic root plaque size was significantly augmented 2.6-fold in PHD2cko, and 1.4-fold in PHD3ko compared to controls, but was unchanged in PHD1ko mice. Macrophage apoptosis was promoted in PHD2cko and PHD3ko mice in vitro and in vivo, via the HIF1α/BNIP3 axis. Bulk and single cell RNA data of PHD2cko bone-marrow-derived macrophages (BMDM) and plaque macrophages, respectively, showed enhanced HIF1α/BNIP3 signaling, which was validated in vitro by siRNA silencing. Human plaque BNIP3 mRNA was positively associated with plaque necrotic core size, suggesting similar pro-apoptotic effects in human. Further, PHD2cko plaques displayed enhanced fibrosis, while macrophage collagen breakdown by matrix metalloproteinases, collagen production and proliferation were unaltered. Instead, PHD2cko BMDMs enhanced fibroblast collagen secretion in a paracrine manner. In silico analysis of macrophage-fibroblast communication predicted SPP1 (osteopontin) signaling as regulator, which was corroborated by enhanced plaque SPP1 protein in vivo. Increased SPP1 mRNA expression upon PHD2cko was preferentially observed in foamy plaque macrophages expressing “triggering receptor expressed on myeloid cells-2” (TREM2hi) evidenced by single-cell RNA, but not in neutrophils. This confirmed enhanced fibrotic signaling by PHD2cko macrophages to fibroblasts, in vitro as well as in vivo. Conclusion Myeloid PHD2cko and PHD3ko enhanced atherosclerotic plaque growth and macrophage apoptosis, while PHD2cko macrophages further activated collagen secretion by fibroblasts in vitro, likely via paracrine SPP1 signaling through TREM2hi macrophages. TRANSLATIONAL OUTLOOK This study shows that myeloid PHD isoforms PHD2 and PHD3 worsen plaque characteristics and phenotype, such as plaque size, macrophage accumulation, apoptosis, and collagen accumulation in mice. We show both direct effects on macrophages and paracrine effects of macrophage PHD2 loss on vessel wall fibroblast populations. Broad spectrum-PHD inhibitors, e.g. Roxadustat, are currently being prescribed to chronic kidney disease patients, who are already at risk for cardiovascular disease. When considering this study and the pro-fibrotic and pro-apoptotic effects we report, broad PHD inhibition may therefore be sub-optimal and more targeted PHD inhibition of PHD1 should be considered.