Archive 21/6/29

SARS-CoV-2 viral nonstructural proteins NSP1 and NSP13 can block interferon activation, leading to escaping from innate immune attacks

A group from University of Pennsylvania Perelman School of Medicine, USA, etc. has reported that SARS-CoV-2 viral nonstructural proteins NSP1 and NSP13 can block interferon activation via distinct mechanisms.

The innate immune antiviral activity provides the first barrier of protection against viral pathogens. Upon RNA virus infection, viral pathogen associated molecular patterns such as viral RNA, spike protein are sensed by cytosolic pattern recognition receptors including RIG-I, MDA5, and TLR family members. RIG-I-viral RNA binding initiates a downstream signaling cascade through a central innate immune adaptor protein, Mitochondrial antiviral-signaling protein (MAVS). Once MAVS is activated, the kinases Tank-binding kinase1 (TBK1) and I-kappa-B epsilon kinase (IKKε) phosphorylate Interferon Regulatory Factor 3 (IRF3), and leads to its nuclear translocation and transcriptional induction of interferon-beta (IFN-β). Secreted IFN-β binds to the IFN-ɑ/β receptors (IFNAR) on neighboring cells, and initiates the interferon response and signaling through the JAK/STAT receptor kinases to produce interferon-stimulated genes (ISGs).

It was shown that SARS-CoV-2 viral nonstructural proteins NSP1 and NSP13 limit IFN response promoter and NF-κB activation, especially NSP1 inhibits nascent host translation and NSP13 interacts with TBK1 and blocks TBK1-mediated IFN activation.

Roles of ΔH69/V70 mutation seen in multiple independent lineages of SARS-CoV-2 

A group from University of Cambridge, UK, etc. has reported on roles of ΔH69/V70 mutation seen in multiple independent lineages of SARS-CoV-2.

There was no overall change in susceptibility to serum neutralization for ΔH69/V70 relative to the WT, suggesting that ΔH69/V70 does not represent an important antibody escape mechanism.

Western blotting for S2 spike indicated a higher amount of cleaved spike in ΔH69/V70 bearing virions and in the HEK293T producer cell lysates. 

As a conclusion, it was found that ΔH69/V70 itself is not an antibody evasion mechanism, but increases infectivity associated with enhanced incorporation of cleaved spike into virions.

HLA-I and HLA-II antigens derived from SARS-CoV-2 

A group from Weizmann Institute of Science, Rehovot, Israel, etc. has reported on HLA-I and HLA-II antigens derived from SARS-CoV-2.

HEK293T cell line stably expressing angiotensin-converting enzyme 2 (ACE2) and IHW01070, Calu-3 cell lines were infected with SARS-CoV-2. The, HLA peptidomics to profile HLA-I- and HLA-II-bound antigens was performed. All HLA peptidomics analyses were performed in triplicate.

We identified 10 different HLA-I peptides derived from canonical ORFs of the virus.
Two peptides were derived from the spike protein, NEVAKNLNESL and TGSNVFQTR,
Two peptides were derived from nsp3, STTTNIVTR and YYTSNPTTF,
One peptide was derived from ORF3, FTIGTVTLK,
One peptide was derived from nsp1, HSSGVTREL,
Four peptides were derived from nucleocapsid, APRITFGGP, RITFGGPSD, NAPRITFGGP and ITFGGPSDSTGSNQNGER.

A nested set of 25 peptides as HLA-II peptides was derived from membrane as shown below.

These information will help to develop vaccines.

Fc-mediated effector functions and Antibody-Dependent Enhancement (ADE): a potent SARS-CoV-2 neutralizing antibody

A group from Biological Defense Program, DSO National Laboratories, Singapore, etc. has reported on Fc-mediated effector functions of a potent SARS-CoV-2 neutralizing antibody.

Antibody-Dependent Enhancement (ADE) of disease remains a major concern for the use of anti-SARS-CoV-2 antibodies as therapeutics. ADE can occur if Fcγ Receptor (FcγR) engagement mediates an increase in the infection of phagocytic cells. Due to the potential of ADE, several ongoing SARS-CoV-2 antibody programs have chosen to use Fc isotypes that do not engage FcγR, like the IgG4 isotype, and engineered variants such as those carrying FcγR-null LALA variant. However, these may be counterproductive because the signaling mechanisms underpinning the efficacies of these antibodies, particularly the ability of FcγR engagement to induce other antiviral responses such as ADCC will be killed. To address these questions, authors isolated and characterized a RBD-binding neutralizing IgG1 antibody, named SC31, from an early convalescent patient. Authors assessed the impact of Fc functionality on its therapeutic efficacy by comparing SC31 with its LALA variant and demonstrated that the engagement of Fc receptors by SC31 triggers additional IFN-γ-mediated antiviral responses but importantly do not induce ADE.

To determine the role of Fc-mediated effector functions in the therapeutic efficacy of SC31, the abilities of SC31 and its LALA variant were compared. The upstream activation of the FcγRIIIa ADCC signalling pathway was evaluated using a Jurkat reporter cell line expressing FcγRIIIa and with ADCC reporter assay after co-culture with target HEK293 cells expressing membrane-bound SARS-CoV-2 Spike protein. In contrast to its LALA variant, SC31 was confirmed to induce a dose-dependent activation of ADCC signaling.

50-gene risk profiles in peripheral blood associated with COVID-19 disease severity 

A group from University of South Florida, Morsani College of Medicine, USA, etc. has reported 50-gene risk profiles in peripheral blood associated with COVID-19 disease severity.

In high risk groups, 7 genes were up-regulated (PLBD1, TPST1, MCEMP1, IL1R2, HP, FLT3, S100A12), and 43 genes were down-regulated (LCK, CAMK2D, NUP43, SLAMF7, LRRC39, ICOS, CD47, LBH, SH2D1A, CNOT6L, METTL8, ETS1, P2RY10, TRAT1, BTN3A1, LARP4, TC2N, GPR183, MORC4, STAT4, LPAR6, CPED1, DOCK10, ARHGAP5, HLA-DPA1, BIRC3, GPR174, CD28, UTRN, CD2, HLA-DPB1, ARL4C, BTN3A3, CXCR6, DYNC2LI1, BTN3A2, ITK, CD96, GBP4, S1PR1, NAP1L2, KLF12, IL7R).

A list of 50-gene expressing cells are summarized in a Table below. This table provides evidence of the cellular source of 50-gene expression changes in peripheral blood and point at specific cell types potentially associated with increased risk of mortality in COVID-19. It is shown that the increased proportion of CD4+ and CD8+ T lymphocytes and immunoglobulin-producing plasmablasts are related to low-risk groups, suggesting that a strong T cell response is associated with better disease outcomes.

Galectin-3 could be a good marker for Abdominal Aortic Aneurysm

A group from Taipei Medical University, Taiwan, etc. has reported that Galectin-3 could be a good marker for Abdominal Aortic Aneurysm (AAA).

Although ultrasound is the gold standard for the diagnosis and surveillance of AAAs with high sensitivity and specificity, the frequency of ultrasound surveillance varies with aneurysm diameter. Furthermore, ultrasound is not recommended for patients with subaneurysmal aortic dilatation. Therefore, circulating biomarkers of inflammation, which reflect the aneurysmal size, can assist in the detection and prognosis of AAA.

A cross-sectional study was performed to analyze plasma Gal-3 and IL-6 levels as circulating biomarkers in both control patients (n = 195) and patients with AAA (n = 151). Plasma Gal-3 levels were significantly higher in patients with AAA than in control patients (96.9 ± 4.5L vs. 76.5 ± 1.9 ng/mL), and the levels of IL-6 were also higher in AAA samples than in healthy control samples (92.8 ± 5.2 pg/mL vs. 72.5 ± 3.0 pg/mL). The diagnostic performance of Gal-3 and IL-6 were evaluated using ROC analysis. The results were that Gal-3 levels predicted AAA presence (AUC=0.91) significantly more accurately than did IL-6 levels (AUC=0.72).

Gal-3 is likely a chemotactic molecule for macrophages. Thus, its expression could be associated with various cardiovascular diseases. The increased risk of AAA observed in patients with higher Gal-3 levels may reflect the recruitment of inflammatory cells, including activated macrophages, in the arterial system and the subsequent secretion of Gal-3.


Binding specificities of C-type lectins, MBP, Langerin, and Dectin-2, as a summary 

A group from Tohoku Medical and Pharmaceutical University has reviewed 11 kinds of mammalian lectins responsible for pathogen recognition.

From this review, three C-type lectins were highlighted here for your information.
Many C-type lectins show selectivity toward mannose residues and this mannose binding is utilized for microbe sensing. One of the most studied examples is mannose-binding protein, MBP, which is known to activate the complement lectin pathway. Why does mannose recognition play this role in detecting non-self when mannose residues occur frequently in mammalian N-glycans, e.g., high-mannose glycan. The likely explanation is in the higher spatial density of mannose residues on bacteria relative to that of mammalian glycans. The C-type lectin domain has a Ca2+ ion coordinated with the OH3 and OH4 of the mannose. The affinity of 1:1 binding is weak with a dissociation constant of roughly 1 mM, however, the presentation of trimeric binding sites in MBP domains could interact with the multiple terminal mannose residues presented on microbes. The spacing between the mannose binding sites is around 50 Å, and is eminently suitable for binding packed terminal mannose residues with high affinity, but not single endogenous high mannose glycans. Langerin is also a C-type lectin with a coiled-coil region and a neck region in a trimeric structure, and has a distance of roughly 40 Å between binding sites. A trimeric oligosaccharide ligand with appropriate linker length for the 40 Å distance between each binding site has been reported for Langerin, with 1,000-fold higher affinity over the monomeric ligand.

As for Dectin-2, the binding site accommodates internally positioned Manα1-2Man of mannans and other polysaccharides, whereas other C-type lectins like DC-SIGN and langerin bind only terminal Manα1-2Man structures. Recognition of internal mannose residue is advantageous in that multiple binding sites are presented toward lectin receptor. Dectin-2 is thus suitable for binding to longer mannan polysaccharides.

As a therapeutic drug for COVID-19,  a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitor was discovered

A group from Washington University School of Medicine, Saint Louis, Missouri, United States has discovered a novel class of small molecule ketobenzothiazole TMPRSS2 inhibitors with significantly improved activity over existing irreversible inhibitors Camostat and Nafamostat. Lead compound MM3122 has an IC50 of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells.

Relationship between serum uric acid level and COVID-19 severity 

A group from Cliniques universitaires Saint-Luc, Belgium, etc. has reported relationship between serum uric acid level and COVID-19 severity.

The prevalence of hypouricemia is approximately 0.3% in the general ambulatory population. In this study, 20% of the patients hospitalized for SARS-CoV-2 infection developed hypouricemia, a proportion that increased to 77% among patients requiring mechanical ventilation.

Uric acid is the end-product of purine metabolism in humans and is generated in the liver. The kidney is an important regulator of circulating uric acid levels as it excretes most of total body uric acid. Serum urate is freely filtered by the glomeruli followed by a complex balance of reabsorption and secretion in the kidney proximal tubule. Although the molecular mechanisms of urate transport in the proximal tubule are still incompletely understood, URAT1 (SLC22A12) is the main apical transporter mediating urate reabsorption in the brush border of the proximal tubule. In a small subset of kidney samples from patients who died from COVID-19, it was shown that life-threatening SARS-CoV-2 infection is associated with a significant (~ 70%) decrease in the expression of the apical urate transporter URAT1 in the kidney proximal tubule, contributing to the impaired tubular absorption of urate. The mechanisms linking hypouricemia and progression to severe disease requiring mechanical ventilation in patients with COVID-19 remains speculative and may be diverse

Anyhow, it seems that serum uric acid could be used as a reliable biomarker to identify patients at risk of life-threatening COVID-19.

Whether HIV antibodies could neutralize SARS-CoV-2 or not 

A group from University of British Columbia, Canada has reported whether HIV antibodies could neutralize SARS-CoV-2 or not.

Cross-reactive interactions of three HIV antibodies (2G12, PGT128, PGT126) in the presence of methyl α-d-mannopyranoside, a stabilized mannose analogue, were evaluated using an ELISA assay. Disruption of cross-reactivity was observed for all these antibodies with increasing concentrations of methyl α-d-mannopyranoside, demonstrating that the binding of these antibodies to SARS-CoV-2 were via glycan sensitivity of these interactions.

HEK293-T cells stably overexpressing ACE-2 were incubated with SARS-CoV-2 S pseudo-typed virus harbouring a luciferase reporter gene, in the presence of serial dilutions of three HIV antibodies. Luciferase activities in cellular lysates were determined 48 h post-infection (RLU: relative luciferase units). However, no neutralization capabilities were detected for these antibodies over a wide range of concentrations, while VH-FC ab8 demonstrated potent neutralization, a positive control antibody VH-FC ab8 which targets the SARS-CoV-2 RBD.

But, the blog admin thinks that the infection to immune cells (i.e., macrophages) expressing C-type lectins might be neutralized by these HIV antibodies? 

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