CDetection.v2: One-pot assay for the detection of SARS-CoV-2.

Introduction: The ongoing 2019 coronavirus disease pandemic (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, is a global public health threat. Early diagnosis and identification of SARS-CoV-2 and its variants plays a critical role in COVID-19 prevention and control. Currently, the most widely used technique to detect SARS-CoV-2 is quantitative reverse transcription real-time quantitative PCR (RT-qPCR), which takes nearly 1 hour and should be performed by experienced personnel to ensure the accuracy of results. Therefore, the development of a nucleic acid detection kit with higher sensitivity, faster detection and greater accuracy is important. Methods: Here, we optimized the system components and reaction conditions of our previous detection approach by using RT-RAA and Cas12b. Results: We developed a Cas12b-assisted one-pot detection platform (CDetection.v2) that allows rapid detection of SARS-CoV-2 in 30 minutes. This platform was able to detect up to 5,000 copies/ml of SARS-CoV-2 without cross-reactivity with other viruses. Moreover, the sensitivity of this CRISPR system was comparable to that of RT-qPCR when tested on 120 clinical samples. Discussion: The CDetection.v2 provides a novel one-pot detection approach based on the integration of RT-RAA and CRISPR/Cas12b for detecting SARS-CoV-2 and screening of large-scale clinical samples, offering a more efficient strategy for detecting various types of viruses.

Dendric nanostructures for SARS-CoV-2 proteins detection based on surface enhanced infrared absorption spectroscopy.

Infrared spectroscopy is a non-destructive and rapid characterization tool that can distinguish different viral proteins by spectral details. However, traditional infrared spectroscopy has insufficient absorption signal intensity contrast when measuring low-concentration samples. In this work, surface enhanced infrared absorption (SEIRA) spectroscopy is proposed by deploying a novel nanostructure array as SEIRA substrates. An array of gold dendric nanostructures are designed and fabricated with a precision resonance control to achieve surface enhancement covering a broadband molecular "finger-print" region. The spectral positions of the multiple resonances accurately correspond to the characteristic absorption peaks of the SARS-CoV-2 proteins. An approach for SARS-CoV-2 protein detection based on SEIRA spectroscopy is then proposed. A low concentration detection of 40 µg/ml diluted SARS-CoV-2 nucleocapsid protein is experimentally demonstrated and the enhancement factor (EF) achieved is in good agreement with simulation results. The SEIRA methodology based on broadband resonance nanostructure design provides a systematic approach for sensitive, non-destructive and rapid protein molecular detection, which could be extended to various kind of molecular characterization and biomedical diagnostics.

Landscape of Peripheral Blood Mononuclear Cells and Soluble Factors in Severe COVID-19 Patients With Pulmonary Fibrosis Development.

Resulting from severe inflammation and cell destruction, COVID-19 patients could develop pulmonary fibrosis (PF), which remains in the convalescent stage. Nevertheless, how immune response participates in the pathogenesis of PF progression is not well defined. To investigate that question, 12 patients with severe COVID-19 were included in the study. Peripheral mononuclear cell (PBMC) samples were collected shortly after their admission and proceeded for single-cell RNA sequencing (scRNA-seq). After 14 days of discharge, the patients were revisited for chest CT scan. PF index (FI) was computed by AI-assisted CT images. Patients were categorized into FIhi and FIlo based on median of FI. By scRNA-seq analysis, our data demonstrated that frequency of CD4+ activated T cells and Treg cells were approximately 3-fold higher in FIhi patients compared with FIlo ones (p < 0.034 for all). By dissecting the differentially expressed genes, we found an overall downregulation of IFN-responsive genes (STAT1, IRF7, ISG15, ISG20, IFIs, and IFITMs) and S100s alarmins (S100A8, S100A9, S100A12, etc.) in all T-cell clusters, and cytotoxicity-related genes (GZMB, PRF1, and GNLY) in CTLs and γδ T cells in the FIhi cohort, compared with FIlo subjects. The GSEA analysis illustrated decreased expression of genes enriched in IFN signaling, innate immune response, adaptive immune response in T cells, NK cells, and monocytes in FIhi patients compared with FIlo ones. In conclusion, these data indicated that the attenuated IFN-responsive genes and their related signaling pathways could be critical for PF progression in COVID-19 patients.

Three doses of an inactivation-based COVID-19 vaccine induces cross-neutralizing immunity against the SARS CoV-2 Omicron variant.

The immunity potency upon natural infection or vaccination is the main concern for the vaccine strategy of severe acute respiratory syndrome coronavirus 2 (SARS COV-2 variant), especially the recently reported Omicron variant (B.1.1.529). In this study, 200 recipients immunized with three doses of a COVID-19-inactivated vaccine were enrolled, whose serum samples were collected within 2 months after the third immunization. The neutralizing activity of sera against the pseudotyped Omicron variant, prototype, and Delta variant was determined. Our results demonstrated that the positive neutralization activity was 95.5% for the Omicron variant, 99.5% for the prototype, and 98.5% for the Delta variant. The geometric mean titers (GMT) for the Omicron variant was 49 and maintained sustained immune levels for 2 months, which decreased by 4.9-fold and 3.0-fold compared with the prototype (GMT, 239) and Delta variant (GMT, 148), respectively. In summary, our study demonstrated that three doses of a COVID-19-inactivated vaccine effectively yielded potent cross-neutralizing activity against the Omicron variant at 2 months after the third vaccination.

Mesenchymal stem cell treatment improves outcome of COVID-19 patients via multiple immunomodulatory mechanisms.

The infusion of coronavirus disease 2019 (COVID-19) patients with mesenchymal stem cells (MSCs) potentially improves clinical symptoms, but the underlying mechanism remains unclear. We conducted a randomized, single-blind, placebo-controlled (29 patients/group) phase II clinical trial to validate previous findings and explore the potential mechanisms. Patients treated with umbilical cord-derived MSCs exhibited a shorter hospital stay (P = 0.0198) and less time required for symptoms remission (P = 0.0194) than those who received placebo. Based on chest images, both severe and critical patients treated with MSCs showed improvement by day 7 (P = 0.0099) and day 21 (P = 0.0084). MSC-treated patients had fewer adverse events. MSC infusion reduced the levels of C-reactive protein, proinflammatory cytokines, and neutrophil extracellular traps (NETs) and promoted the maintenance of SARS-CoV-2-specific antibodies. To explore how MSCs modulate the immune system, we employed single-cell RNA sequencing analysis on peripheral blood. Our analysis identified a novel subpopulation of VNN2+ hematopoietic stem/progenitor-like (HSPC-like) cells expressing CSF3R and PTPRE that were mobilized following MSC infusion. Genes encoding chemotaxis factors - CX3CR1 and L-selectin - were upregulated in various immune cells. MSC treatment also regulated B cell subsets and increased the expression of costimulatory CD28 in T cells in vivo and in vitro. In addition, an in vivo mouse study confirmed that MSCs suppressed NET release and reduced venous thrombosis by upregulating kindlin-3 signaling. Together, our results underscore the role of MSCs in improving COVID-19 patient outcomes via maintenance of immune homeostasis.

Different Profiles of Antibodies and Cytokines Were Found Between Severe and Moderate COVID-19 Patients.

Objectives: Our objective was to determine the antibody and cytokine profiles in different COVID-19 patients. Methods: COVID-19 patients with different clinical classifications were enrolled in this study. The level of IgG antibodies, IgA, IgM, IgE, and IgG subclasses targeting N and S proteins were tested using ELISA. Neutralizing antibody titers were determined by using a toxin neutralization assay (TNA) with live SARS-CoV-2. The concentrations of 8 cytokines, including IL-2, IL-4, IL-6, IL-10, CCL2, CXCL10, IFN-γ, and TNF-α, were measured using the Protein Sample Ella-Simple ELISA system. The differences in antibodies and cytokines between severe and moderate patients were compared by t-tests or Mann-Whitney tests. Results: A total of 79 COVID-19 patients, including 49 moderate patients and 30 severe patients, were enrolled. Compared with those in moderate patients, neutralizing antibody and IgG-S antibody titers in severe patients were significantly higher. The concentration of IgG-N antibody was significantly higher than that of IgG-S antibody in COVID-19 patients. There was a significant difference in the distribution of IgG subclass antibodies between moderate patients and severe patients. The positive ratio of anti-S protein IgG3 is significantly more than anti-N protein IgG3, while the anti-S protein IgG4 positive rate is significantly less than the anti-N protein IgG4 positive rate. IL-2 was lower in COVID-19 patients than in healthy individuals, while IL-4, IL-6, CCL2, IFN-γ, and TNF-α were higher in COVID-19 patients than in healthy individuals. IL-6 was significantly higher in severe patients than in moderate patients. The antibody level of anti-S protein was positively correlated with the titer of neutralizing antibody, but there was no relationship between cytokines and neutralizing antibody. Conclusions: Our findings show the severe COVID-19 patients' antibody levels were stronger than those of moderate patients, and a cytokine storm is associated with COVID-19 severity. There was a difference in immunoglobulin type between anti-S protein antibodies and anti-N protein antibodies in COVID-19 patients. And clarified the value of the profile in critical prevention.

A Nomogram Prediction of Length of Hospital Stay in Patients with COVID-19 Pneumonia: A Retrospective Cohort Study.

Assessing the length of hospital stay (LOS) in patients with coronavirus disease 2019 (COVID-19) pneumonia is helpful in optimizing the use efficiency of hospital beds and medical resources and relieving medical resource shortages. This retrospective cohort study of 97 patients was conducted at Beijing You'An Hospital between January 21, 2020, and March 21, 2020. A multivariate Cox proportional hazards regression based on the smallest Akaike information criterion value was used to select demographic and clinical variables to construct a nomogram. Discrimination, area under the receiver operating characteristic curve (AUC), calibration, and Kaplan-Meier curves with the log-rank test were used to assess the nomogram model. The median LOS was 13 days (interquartile range [IQR]: 10-18). Age, alanine aminotransferase, pneumonia, platelet count, and PF ratio (PaO2/FiO2) were included in the final model. The C-index of the nomogram was 0.76 (95%confidence interval [CI] = 0.69-0.83), and the AUC was 0.88 (95%CI = 0.82-0.95). The adjusted C-index was 0.75 (95%CI = 0.67-0.82) and adjusted AUC 0.86 (95%CI = 0.73-0.95), both after 1000 bootstrap cross internal validations. A Brier score of 0.11 (95%CI = 0.07-0.15) and adjusted Brier score of 0.130 (95%CI = 0.07-0.20) for the calibration curve showed good agreement. The AUC values for the nomogram at LOS of 10, 20, and 30 days were 0.79 (95%CI = 0.69-0.89), 0.89 (95%CI = 0.83-0.96), and 0.96 (95%CI = 0.92-1.00), respectively, and the high fit score of the nomogram model indicated a high probability of hospital stay. These results confirmed that the nomogram model accurately predicted the LOS of patients with COVID-19. We developed and validated a nomogram that incorporated five independent predictors of LOS. If validated in a future large cohort study, the model may help to optimize discharge strategies and, thus, shorten LOS in patients with COVID-19.

Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge during the global pandemic and may facilitate escape from current antibody therapies and vaccine protection. Here we showed that the South African variant B.1.351 was the most resistant to current monoclonal antibodies and convalescent plasma from coronavirus disease 2019 (COVID-19)-infected individuals, followed by the Brazilian variant P.1 and the United Kingdom variant B.1.1.7. This resistance hierarchy corresponded with Y144del and 242-244del mutations in the N-terminal domain and K417N/T, E484K, and N501Y mutations in the receptor-binding domain (RBD) of SARS-CoV-2. Crystal structure analysis of the B.1.351 triple mutant (417N-484K-501Y) RBD complexed with the monoclonal antibody P2C-1F11 revealed the molecular basis for antibody neutralization and escape. B.1.351 and P.1 also acquired the ability to use mouse and mink ACE2 receptors for entry. Our results demonstrate major antigenic shifts and potential broadening of the host range for B.1.351 and P.1 variants, which poses serious challenges to current antibody therapies and vaccine protection.

Humoral immune response to circulating SARS-CoV-2 variants elicited by inactivated and RBD-subunit vaccines.

SARS-CoV-2 variants could induce immune escape by mutations on the receptor-binding domain (RBD) and N-terminal domain (NTD). Here we report the humoral immune response to circulating SARS-CoV-2 variants, such as 501Y.V2 (B.1.351), of the plasma and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine), ZF2001 (RBD-subunit vaccine) and natural infection. Among 86 potent NAbs identified by high-throughput single-cell VDJ sequencing of peripheral blood mononuclear cells from vaccinees and convalescents, near half anti-RBD NAbs showed major neutralization reductions against the K417N/E484K/N501Y mutation combination, with E484K being the dominant cause. VH3-53/VH3-66 recurrent antibodies respond differently to RBD variants, and K417N compromises the majority of neutralizing activity through reduced polar contacts with complementarity determining regions. In contrast, the 242-244 deletion (242-244Δ) would abolish most neutralization activity of anti-NTD NAbs by interrupting the conformation of NTD antigenic supersite, indicating a much less diversity of anti-NTD NAbs than anti-RBD NAbs. Plasma of convalescents and CoronaVac vaccinees displayed comparable neutralization reductions against pseudo- and authentic 501Y.V2 variants, mainly caused by E484K/N501Y and 242-244Δ, with the effects being additive. Importantly, RBD-subunit vaccinees exhibit markedly higher tolerance to 501Y.V2 than convalescents, since the elicited anti-RBD NAbs display a high diversity and are unaffected by NTD mutations. Moreover, an extended gap between the third and second doses of ZF2001 leads to better neutralizing activity and tolerance to 501Y.V2 than the standard three-dose administration. Together, these results suggest that the deployment of RBD-vaccines, through a third-dose boost, may be ideal for combating SARS-CoV-2 variants when necessary, especially for those carrying mutations that disrupt the NTD supersite.

Longitudinal Profiling of Antibody Response in Patients With COVID-19 in a Tertiary Care Hospital in Beijing, China.

The novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic of the coronavirus disease 2019 (COVID-19), which elicits a wide variety of symptoms, ranging from mild to severe, with the potential to lead to death. Although used as the standard method to screen patients for SARS-CoV-2 infection, real-time PCR has challenges in dealing with asymptomatic patients and those with an undetectable viral load. Serological tests are therefore considered potent diagnostic tools to complement real-time PCR-based diagnosis and are used for surveillance of seroprevalence in populations. However, the dynamics of the antibody response against SARS-CoV-2 currently remain to be investigated. Here, through analysis of plasma samples from 84 patients with COVID-19, we observed that the response of virus-specific antibodies against three important antigens, RBD, N and S, dynamically changed over time and reached a peak 5-8 weeks after the onset of symptoms. The antibody responses were irrespective of sex. Severe cases were found to have higher levels of antibody response, larger numbers of inflammatory cells and C-reactive protein levels. Within the mild/moderate cases, pairwise comparison indicated moderate association between anti-RBD vs. anti-N, anti-RBD vs. anti-S1S2, and anti-N vs. anti-S1S2. Furthermore, the majority of cases could achieve IgM and IgG seroconversion at 2 weeks since the disease onset. Analysis of neutralizing antibodies indicated that these responses were able to last for more than 112 days but decline significantly after the peak. In summary, our findings demonstrate the longitudinally dynamic changes in antibody responses against SARS-CoV-2, which can contribute to the knowledge of humoral immune response after SARS-CoV-2 infection and are informative for future development of vaccine and antibody-based therapies.

Early Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Antibodies as a Serologic Marker of Infection in Patients With Coronavirus Disease 2019.

BACKGROUND: Thousands of medical staff have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with hundreds of deaths reported. Such loss could be prevented if there were a serologic assay for SARS-CoV-2-specific antibodies for serological surveillance of its infection at the early stage of disease. METHODS: Using Chinese hamster ovarian (CHO) cell-expressed full-length SARS-CoV-2 S1 protein as capturing antigen, a coronavirus disease 2019 (COVID-19)/SARS-CoV-2 S1 serology enzyme-linked immunosorbent assay (ELISA) kit was developed and validated with negative samples collected prior to the outbreak or during the outbreak and positive samples from patients confirmed with COVID-19. RESULTS: The specificity of the ELISA kit was 97.5%, as examined against total 412 normal human samples. The sensitivity was 97.1% by testing against 69 samples from hospitalized and/or recovered COVID-19 patients. The overall accuracy rate reached 97.3%. The assay was able to detect SARS-CoV-2 antibody on day 1 after the onset of COVID-19 disease. The average antibody levels increased during hospitalization and 14 days after discharge. SARS-CoV-2 antibodies were detected in 28 of 276 asymptomatic medical staff and 1 of 5 nucleic acid test-negative "close contacts" of COVID-19 patients. CONCLUSIONS: With the assays developed here, we can screen medical staff, incoming patients, passengers, and people who are in close contact with the confirmed patients to identify the "innocent viral spreaders," protect the medical staff, and stop further spread of the virus.

Downregulated Gene Expression Spectrum and Immune Responses Changed During the Disease Progression in Patients With COVID-19.

BACKGROUND: The World Health Organization characterizes novel coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), as a pandemic. Here, we investigated the clinical, cytokine levels; T-cell proportion; and related gene expression occurring in patients with COVID-19 on admission and after initial treatment. METHODS: Eleven patients diagnosed with COVID-19 with similar initial treatment regimens were enrolled in the hospital. Plasma cytokine, peripheral T cell proportions, and microfluidic quantitative polymerase chain reaction analyses for gene expression were conducted. RESULTS: Five patients with mild and 6 with severe disease were included. Cough and fever were the primary symptoms in the 11 COVID-19 cases. Older age, higher neutrophil count, and higher C-reactive protein levels were found in severe cases. IL-10 level significantly varied with disease progression and treatment. Decreased T-cell proportions were observed in patients with COVID-19, especially in severe cases, and all were returned to normal in patients with mild disease after initial treatment, but only CD4+ T cells returned to normal in severe cases. The number of differentially expressed genes (DEGs) increased with the disease progression, and decreased after initial treatment. All downregulated DEGs in severe cases mainly involved Th17-cell differentiation, cytokine-mediated signaling pathways, and T-cell activation. After initial treatment in severe cases, MAP2K7 and SOS1 were upregulated relative to that on admission. CONCLUSIONS: Our findings show that a decreased T-cell proportion with downregulated gene expression related to T-cell activation and differentiation occurred in patients with severe COVID-19, which may help to provide effective treatment strategies for COVID-19.

Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development.

To discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

Transplantation of ACE2- Mesenchymal Stem Cells Improves the Outcome of Patients with COVID-19 Pneumonia.

A coronavirus (HCoV-19) has caused the novel coronavirus disease (COVID-19) outbreak in Wuhan, China. Preventing and reversing the cytokine storm may be the key to save the patients with severe COVID-19 pneumonia. Mesenchymal stem cells (MSCs) have been shown to possess a comprehensive powerful immunomodulatory function. This study aims to investigate whether MSC transplantation improves the outcome of 7 enrolled patients with COVID-19 pneumonia in Beijing YouAn Hospital, China, from Jan 23, 2020 to Feb 16, 2020. The clinical outcomes, as well as changes of inflammatory and immune function levels and adverse effects of 7 enrolled patients were assessed for 14 days after MSC injection. MSCs could cure or significantly improve the functional outcomes of seven patients without observed adverse effects. The pulmonary function and symptoms of these seven patients were significantly improved in 2 days after MSC transplantation. Among them, two common and one severe patient were recovered and discharged in 10 days after treatment. After treatment, the peripheral lymphocytes were increased, the C-reactive protein decreased, and the overactivated cytokine-secreting immune cells CXCR3+CD4+ T cells, CXCR3+CD8+ T cells, and CXCR3+ NK cells disappeared in 3-6 days. In addition, a group of CD14+CD11c+CD11bmid regulatory DC cell population dramatically increased. Meanwhile, the level of TNF-α was significantly decreased, while IL-10 increased in MSC treatment group compared to the placebo control group. Furthermore, the gene expression profile showed MSCs were ACE2- and TMPRSS2- which indicated MSCs are free from COVID-19 infection. Thus, the intravenous transplantation of MSCs was safe and effective for treatment in patients with COVID-19 pneumonia, especially for the patients in critically severe condition.

COVID-19 immune features revealed by a large-scale single-cell transcriptome atlas.

A dysfunctional immune response in coronavirus disease 2019 (COVID-19) patients is a recurrent theme impacting symptoms and mortality, yet a detailed understanding of pertinent immune cells is not complete. We applied single-cell RNA sequencing to 284 samples from 196 COVID-19 patients and controls and created a comprehensive immune landscape with 1.46 million cells. The large dataset enabled us to identify that different peripheral immune subtype changes are associated with distinct clinical features, including age, sex, severity, and disease stages of COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA was found in diverse epithelial and immune cell types, accompanied by dramatic transcriptomic changes within virus-positive cells. Systemic upregulation of S100A8/A9, mainly by megakaryocytes and monocytes in the peripheral blood, may contribute to the cytokine storms frequently observed in severe patients. Our data provide a rich resource for understanding the pathogenesis of and developing effective therapeutic strategies for COVID-19.

Neutralizing Antibody Responses to Severe Acute Respiratory Syndrome Coronavirus 2 in Coronavirus Disease 2019 Inpatients and Convalescent Patients.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a pandemic with no specific antiviral treatments or vaccines. There is an urgent need for exploring the neutralizing antibodies from patients with different clinical characteristics. METHODS: A total of 117 blood samples were collected from 70 COVID-19 inpatients and convalescent patients. Antibodies were determined with a modified cytopathogenic neutralization assay (NA) based on live severe acute respiratory syndrome coronavirus 2 and enzyme-linked immunosorbent assay (ELISA). The dynamics of neutralizing antibody levels at different time points with different clinical characteristics were analyzed. RESULTS: The seropositivity rate reached up to 100.0% within 20 days since onset, and remained 100.0% till days 41-53. The total geometric mean titer was 1:163.7 (95% confidence interval [CI], 128.5-208.6) by NA and 1:12 441.7 (95% CI, 9754.5-15 869.2) by ELISA. The antibody level by NA and ELISA peaked on days 31-40 since onset, and then decreased slightly. In multivariate generalized estimating equation analysis, patients aged 31-45, 46-60, and 61-84 years had a higher neutralizing antibody level than those aged 16-30 years (ß = 1.0470, P = .0125; ß = 1.0613, P = .0307; ß = 1.3713, P = .0020). Patients with a worse clinical classification had a higher neutralizing antibody titer (ß = 0.4639, P = .0227). CONCLUSIONS: The neutralizing antibodies were detected even at the early stage of disease, and a significant response was shown in convalescent patients.