Association of complement pathways with COVID-19 severity and outcomes.

OBJECTIVES: Complement activation has been implicated in COVID-19 pathogenesis. This study aimed to assess the levels of complement activation products and full-length proteins in hospitalized patients with COVID-19, and evaluated whether complement pathway markers are associated with outcomes. METHODS: Longitudinal measurements of complement biomarkers from 89 hospitalized adult patients, grouped by baseline disease severity, enrolled in an adaptive, phase 2/3, randomized, double-blind, placebo-controlled trial and treated with intravenous sarilumab (200 mg or 400 mg) or placebo (NCT04315298), were performed. These measurements were then correlated with clinical and laboratory parameters. RESULTS: All complement pathways were activated in hospitalized patients with COVID-19. Alternative pathway activation was predominant earlier in the disease course. Complement biomarkers correlated with multiple variables of multi-organ dysfunction and inflammatory injury. High plasma sC5b-9, C3a, factor Bb levels, and low mannan-binding lectin levels were associated with increased mortality. Sarilumab treatment showed a modest inhibitory effect on complement activation. Moreover, sera from patients spontaneously deposited C5b-9 complex on the endothelial surface ex vivo, suggesting a microvascular thrombotic potential. CONCLUSION: These results advance our understanding of COVID-19 disease pathophysiology and demonstrate the importance of specific complement pathway components as prognostic biomarkers in COVID-19.

An Intelligent Graphene-Based Biosensing Device for Cytokine Storm Syndrome Biomarkers Detection in Human Biofluids.

Abnormal elevated levels of cytokines such as interferon (IFN), interleukin (IL), and tumor necrosis factor (TNF), are considered as one of the prognosis biomarkers for indicating the progression to severe or critical COVID-19. Hence, it is of great significance to develop devices for monitoring their levels in COVID-19 patients, and thus enabling detecting COVID-19 patients that are worsening and to treat them before they become critically ill. Here, an intelligent aptameric dual channel graphene-TWEEN 80 field effect transistor (DGTFET) biosensing device for on-site detection of IFN-γ, TNF-α, and IL-6 within 7 min with limits of detection (LODs) of 476 × 10-15 , 608 × 10-15 , or 611 × 10-15 m respectively in biofluids is presented. Using the customized Android App together with this intelligent device, asymptomatic or mild COVID-19 patients can have a preliminary self-detection of cytokines and get a warning reminder while the condition starts to deteriorate. Also, the device can be fabricated on flexible substrates toward wearable applications for moderate or even critical COVID-19 cases for consistently monitoring cytokines under different deformations. Hence, the intelligent aptameric DGTFET biosensing device is promising to be used for point-of-care applications for monitoring conditions of COVID-19 patients who are in different situations.

Neutralization sensitivity, fusogenicity, and infectivity of Omicron subvariants.

BACKGROUND: The emergence of SARS-CoV-2 Omicron subvariants has raised questions regarding resistance to immunity by natural infection or immunization. We examined the sensitivity of Delta and Omicron subvariants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/5, and BA.3) to neutralizing antibodies from BBIBP-CorV-vaccinated and BBIBP-CorV- or ZF2001-boosted individuals, as well as individuals with Delta and BA.1 breakthrough infections, and determined their fusogenicity and infectivity. METHODS: In this cross-sectional study, serum samples from two doses of BBIBP-CorV-vaccinated individuals 1 (n = 36), 3 (n = 36), and 7 (n = 37) months after the second dose; BBIBP-CorV- (n = 25) or ZF2001-boosted (n = 30) individuals; and fully vaccinated individuals with Delta (n = 30) or BA.1 (n = 26) infection were collected. The serum-neutralizing reactivity and potency of bebtelovimab were assessed against D614G, Delta, and Omicron subvariants (BA.1, BA.1.1, BA.2, BA.2.12.1, BA.4/5, and BA.3) through a pseudovirus neutralization assay. The fusogenicity and infectivity of D614G, Delta, and Omicron subvariants were determined by cell-cell fusion assay and pseudovirus infection assay, respectively. RESULTS: Omicron subvariants markedly escaped vaccine-elicited neutralizing antibodies after two doses of BBIBP-CorV with comparable efficiency. A third dose vaccination of BBIBP-CorV or ZF2001 increased neutralizing antibody titers and breadth against Delta and three Omicron subvariants. Delta and BA.1 breakthrough infections induced comparable neutralizing antibody titers against D614G and Delta variants, whereas BA.1 breakthrough infections elicited a stronger and broader antibody response against three Omicron subvariants than Delta breakthrough infections. BA.2.12.1 and BA.4/5 are more resistant to immunity induced by breakthrough infections. Bebtelovimab had no significant loss of potency against the Delta and Omicron subvariants. Cell culture experiments showed Omicron subvariants to be less fusogenic and have higher infectivity than D614G and Delta with comparable efficiency. CONCLUSIONS: These findings have important public health implications and highlight the importance of repeated exposure to SARS-CoV-2 antigens to broaden the neutralizing antibody response against Omicron subvariants.

Humanized C3 Mouse: A Novel Accelerated Model of C3 Glomerulopathy.

BACKGROUND: C3 glomerulopathy (C3G) is characterized by the alternative-pathway (AP) hyperactivation induced by nephritic factors or complement gene mutations. Mice deficient in complement factor H (CFH) are a classic C3G model, with kidney disease that requires several months to progress to renal failure. Novel C3G models can further contribute to understanding the mechanism behind this disease and developing therapeutic approaches. METHODS: A novel, rapidly progressing, severe, murine model of C3G was developed by replacing the mouse C3 gene with the human C3 homolog using VelociGene technology. Functional, histologic, molecular, and pharmacologic assays characterize the presentation of renal disease and enable useful pharmacologic interventions in the humanized C3 (C3hu/hu) mice. RESULTS: The C3hu/hu mice exhibit increased morbidity early in life and die by about 5-6 months of age. The C3hu/hu mice display elevated biomarkers of kidney dysfunction, glomerulosclerosis, C3/C5b-9 deposition, and reduced circulating C3 compared with wild-type mice. Administration of a C5-blocking mAb improved survival rate and offered functional and histopathologic benefits. Blockade of AP activation by anti-C3b or CFB mAbs also extended survival and preserved kidney function. CONCLUSIONS: The C3hu/hu mice are a useful model for C3G because they share many pathologic features consistent with the human disease. The C3G phenotype in C3hu/hu mice may originate from a dysregulated interaction of human C3 protein with multiple mouse complement proteins, leading to unregulated C3 activation via AP. The accelerated disease course in C3hu/hu mice may further enable preclinical studies to assess and validate new therapeutics for C3G.

Inhibitory effect on SARS-CoV-2 infection of neferine by blocking Ca2+ -dependent membrane fusion.

The coronavirus disease 2019 (COVID-19) pandemic has focused attention on the need to develop effective therapeutics against the causative pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also against other pathogenic coronaviruses. In this study, we report on a kind of bisbenzylisoquinoline alkaloid, neferine, as a pan-coronavirus entry inhibitor. Neferine effectively protected HEK293/hACE2 and HuH7 cell lines from infection by different coronaviruses pseudovirus particles (SARS-CoV-2, SARS-CoV-2 [D614G, N501Y/D614G, 501Y.V1, 501Y.V2, 501Y.V3 variants], SARS-CoV, MERS-CoV) in vitro, with median effect concentration (EC50 ) of 0.13-0.41 µM. Neferine blocked host calcium channels, thus inhibiting Ca2+ -dependent membrane fusion and suppressing virus entry. This study provides experimental data to support the fact that neferine may be a promising lead for pan-coronaviruses therapeutic drug development.

A Wearable and Deformable Graphene-Based Affinity Nanosensor for Monitoring of Cytokines in Biofluids.

A wearable and deformable graphene-based field-effect transistor biosensor is presented that uses aptamer-modified graphene as the conducting channel, which is capable of the sensitive, consistent and time-resolved detection of cytokines in human biofluids. Based on an ultrathin substrate, the biosensor offers a high level of mechanical durability and consistent sensing responses, while conforming to non-planar surfaces such as the human body and withstanding large deformations (e.g., bending and stretching). Moreover, a nonionic surfactant is employed to minimize the nonspecific adsorption of the biosensor, hence enabling cytokine detection (TNF-α and IFN-γ, significant inflammatory cytokines, are used as representatives) in artificial tears (used as a biofluid representative). The experimental results demonstrate that the biosensor very consistently and sensitively detects TNF-α and IFN-γ, with limits of detection down to 2.75 and 2.89 pM, respectively. The biosensor, which undergoes large deformations, can thus potentially provide a consistent and sensitive detection of cytokines in the human body.

An optimized and robust SARS-CoV-2 pseudovirus system for viral entry research.

SARS-CoV-2 is the culprit causing Coronavirus Disease 2019 (COVID-19). For the study of SARS-CoV-2 infection in a BSL-2 laboratory, a SARS-CoV-2 pseudovirus particle (SARS2pp) production and infection system was constructed by using a lentiviral vector bearing dual-reporter genes eGFP and firefly luciferase (Luc2) for easy observation and analysis. Comparison of SARS2pp different production conditions revealed that the pseudovirus titer could be greatly improved by: 1) removing the last 19 amino acids of the spike protein and replacing the signal peptide with the mouse Igk signal sequence; 2) expressing the spike protein using CMV promoter other than CAG (a hybrid promoter consisting of a CMV enhancer, beta-actin promoter, splice donor, and a beta-globin splice acceptor); 3) screening better optimized spike protein sequences for SARS2pp production; and 4) adding 1 % BSA in the SARS2pp production medium. For infection, this SARS2pp system showed a good linear relationship between MOI 2-0.0002 and then was successfully used to evaluate SARS-CoV-2 infection inhibitors including recombinant human ACE2 proteins and SARS-CoV-2 neutralizing antibodies. The kidney, liver and small intestine-derived cell lines were also found to show different susceptibility to SARSpp and SARS2pp. Given its robustness and good performance, it is believed that this pseudovirus particle production and infection system will greatly promote future research for SARS-CoV-2 entry mechanisms and inhibitors and can be easily applied to study new emerging SARS-CoV-2 variants.