Single-cell analysis of the adaptive immune response to SARS-CoV-2 infection and vaccination.

Amid the ongoing Coronavirus Disease 2019 (COVID-19) pandemic, vaccination and early therapeutic interventions are the most effective means to combat and control the severity of the disease. Host immune responses to SARS-CoV-2 and its variants, particularly adaptive immune responses, should be fully understood to develop improved strategies to implement these measures. Single-cell multi-omic technologies, including flow cytometry, single-cell transcriptomics, and single-cell T-cell receptor (TCR) and B-cell receptor (BCR) profiling, offer a better solution to examine the protective or pathological immune responses and molecular mechanisms associated with SARS-CoV-2 infection, thus providing crucial support for the development of vaccines and therapeutics for COVID-19. Recent reviews have revealed the overall immune landscape of natural SARS-CoV-2 infection, and this review will focus on adaptive immune responses (including T cells and B cells) to SARS-CoV-2 revealed by single-cell multi-omics technologies. In addition, we explore how the single-cell analyses disclose the critical components of immune protection and pathogenesis during SARS-CoV-2 infection through the comparison between the adaptive immune responses induced by natural infection and by vaccination.

Single-cell analysis of the adaptive immune response to SARS-CoV-2 infection and vaccination

Amid the ongoing Coronavirus Disease 2019 (COVID-19) pandemic, vaccination and early therapeutic interventions are the most effective means to combat and control the severity of the disease. Host immune responses to SARS-CoV-2 and its variants, particularly adaptive immune responses, should be fully understood to develop improved strategies to implement these measures. Single-cell multi-omic technologies, including flow cytometry, single-cell transcriptomics, and single-cell T-cell receptor (TCR) and B-cell receptor (BCR) profiling, offer a better solution to examine the protective or pathological immune responses and molecular mechanisms associated with SARS-CoV-2 infection, thus providing crucial support for the development of vaccines and therapeutics for COVID-19. Recent reviews have revealed the overall immune landscape of natural SARS-CoV-2 infection, and this review will focus on adaptive immune responses (including T cells and B cells) to SARS-CoV-2 revealed by single-cell multi-omics technologies. In addition, we explore how the single-cell analyses disclose the critical components of immune protection and pathogenesis during SARS-CoV-2 infection through the comparison between the adaptive immune responses induced by natural infection and by vaccination.

Multiomics approach reveals the ubiquitination-specific processes hijacked by SARS-CoV-2.

The Coronavirus Disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a global pandemic that seriously threatens health and socioeconomic development, but the existed antiviral drugs and vaccines still cannot yet halt the spread of the epidemic. Therefore, a comprehensive and profound understanding of the pathogenesis of SARS-CoV-2 is urgently needed to explore effective therapeutic targets. Here, we conducted a multiomics study of SARS-CoV-2-infected lung epithelial cells, including transcriptomic, proteomic, and ubiquitinomic. Multiomics analysis showed that SARS-CoV-2-infected lung epithelial cells activated strong innate immune response, including interferon and inflammatory responses. Ubiquitinomic further reveals the underlying mechanism of SARS-CoV-2 disrupting the host innate immune response. In addition, SARS-CoV-2 proteins were found to be ubiquitinated during infection despite the fact that SARS-CoV-2 itself didn't code any E3 ligase, and that ubiquitination at three sites on the Spike protein could significantly enhance viral infection. Further screening of the E3 ubiquitin ligases and deubiquitinating enzymes (DUBs) library revealed four E3 ligases influencing SARS-CoV-2 infection, thus providing several new antiviral targets. This multiomics combined with high-throughput screening study reveals that SARS-CoV-2 not only modulates innate immunity, but also promotes viral infection, by hijacking ubiquitination-specific processes, highlighting potential antiviral and anti-inflammation targets.

Structural and functional analysis of an inter-Spike bivalent neutralizing antibody against SARS-CoV-2 variants.

The different variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have attracted most public concern because they caused "wave and wave" COVID-19 pandemic. The initial step of viral infection is mediated by the SARS-CoV-2 Spike (S) protein, which mediates the receptor recognition and membrane fusion between virus and host cells. Neutralizing antibodies (nAbs) targeting the S protein of SARS-CoV-2 have become promising candidates for clinical intervention strategy, while multiple studies have shown that different variants have enhanced infectivity and antibody resistance. Here, we explore the structure and function of STS165, a broadly inter-Spike bivalent nAb against SARS-CoV-2 variants and even SARS-CoV, contributing to further understanding of the working mechanism of nAbs.

The Transient IFN Response and the Delay of Adaptive Immunity Feature the Severity of COVID-19.

COVID-19 patients show heterogeneous and dynamic immune features which determine the clinical outcome. Here, we built a single-cell RNA sequencing (scRNA-seq) dataset for dissecting these complicated immune responses through a longitudinal survey of COVID-19 patients with various categories of outcomes. The data reveals a highly fluctuating peripheral immune landscape in severe COVID-19, whereas the one in asymptomatic/mild COVID-19 is relatively steady. Then, the perturbed immune landscape in peripheral blood returned to normal state in those recovered from severe COVID-19. Importantly, the imbalance of the excessively strong innate immune response and delayed adaptive immunity in the early stage of viral infection accelerates the progression of the disease, indicated by a transient strong IFN response and weak T/B-cell specific response. The proportion of abnormal monocytes appeared early and rose further throughout the severe disease. Our data indicate that a dynamic immune landscape is associated with the progression and recovery of severe COVID-19, and have provided multiple immune biomarkers for early warning of severe COVID-19.

Dysregulated hematopoiesis in bone marrow marks severe COVID-19.

Severe coronavirus disease 2019 (COVID-19) is often indicated by lymphopenia and increased myelopoiesis; however, the underlying mechanism is still unclear, especially the alteration of hematopoiesis. It is important to explore to what extent and how hematopoietic stem cells contribute to the impairment of peripheral lymphoid and myeloid compartments in COVID-19 patients. In this study, we used single-cell RNA sequencing to assess bone marrow mononuclear cells from COVID-19 patients with peripheral blood mononuclear cells as control. The results showed that the hematopoietic stem cells in these patients were mainly in the G1 phase and prone to apoptosis, with immune activation and anti-viral responses. Importantly, a significant accumulation of immature myeloid progenitors and a dramatic reduction of lymphoid progenitors in severe cases were identified, along with the up-regulation of transcription factors (such as SPI1, LMO4, ETS2, FLI1, and GATA2) that are important for the hematopoietic stem cell or multipotent progenitor to differentiate into downstream progenitors. Our results indicate a dysregulated hematopoiesis in patients with severe COVID-19.

Suppressive Monocytes Impair MAIT Cells Response via IL-10 in Patients with Severe COVID-19.

Immune cell responses are strikingly altered in patients with severe coronavirus disease 2019 (COVID-19), but the immunoregulatory process in these individuals is not fully understood. In this study, 23 patients with mild and 22 patients with severe COVID-19 and 6 asymptomatic carriers of COVID-19 were enrolled, along with 44 healthy controls (HC). Peripheral immune cells in HC and patients with COVID-19 were comprehensively profiled using mass cytometry. We found that in patients with severe COVID-19, the number of HLA-DRlow/- monocytes was significantly increased, but that of mucosal-associated invariant T (MAIT) cells was greatly reduced. MAIT cells were highly activated but functionally impaired in response to Escherichia coli and IL-12/IL-18 stimulation in patients with severe COVID-19, especially those with microbial coinfection. Single-cell transcriptome analysis revealed that IFN-stimulated genes were significantly upregulated in peripheral MAIT cells and monocytes from patients with severe COVID-19. IFN-α pretreatment suppressed MAIT cells' response to E. coli by triggering high levels of IL-10 production by HLA-DRlow/--suppressive monocytes. Blocking IFN-α or IL-10 receptors rescued MAIT cell function in patients with severe COVID-19. Moreover, plasma from patients with severe COVID-19 inhibited HLA-DR expression by monocytes through IL-10. These data indicate a unique pattern of immune dysregulation in severe COVID-19, which is characterized by enrichment of suppressive HLA-DRlow/- monocytes associated with functional impairment of MAIT cells through the IFN/IL-10 pathway.

Single-Cell RNA Sequencing Analysis of the Immunometabolic Rewiring and Immunopathogenesis of Coronavirus Disease 2019.

Although immune dysfunction is a key feature of coronavirus disease 2019 (COVID-19), the metabolism-related mechanisms remain elusive. Here, by reanalyzing single-cell RNA sequencing data, we delineated metabolic remodeling in peripheral blood mononuclear cells (PBMCs) to elucidate the metabolic mechanisms that may lead to the progression of severe COVID-19. After scoring the metabolism-related biological processes and signaling pathways, we found that mono-CD14+ cells expressed higher levels of glycolysis-related genes (PKM, LDHA and PKM) and PPP-related genes (PGD and TKT) in severe patients than in mild patients. These genes may contribute to the hyperinflammation in mono-CD14+ cells of patients with severe COVID-19. The mono-CD16+ cell population in COVID-19 patients showed reduced transcription levels of genes related to lysine degradation (NSD1, KMT2E, and SETD2) and elevated transcription levels of genes involved in OXPHOS (ATP6V1B2, ATP5A1, ATP5E, and ATP5B), which may inhibit M2-like polarization. Plasma cells also expressed higher levels of the OXPHOS gene ATP13A3 in COVID-19 patients, which was positively associated with antibody secretion and survival of PCs. Moreover, enhanced glycolysis or OXPHOS was positively associated with the differentiation of memory B cells into plasmablasts or plasma cells. This study comprehensively investigated the metabolic features of peripheral immune cells and revealed that metabolic changes exacerbated inflammation in monocytes and promoted antibody secretion and cell survival in PCs in COVID-19 patients, especially those with severe disease.

The differential immune responses to COVID-19 in peripheral and lung revealed by single-cell RNA sequencing.

Understanding the mechanism that leads to immune dysfunction in severe coronavirus disease 2019 (COVID-19) is crucial for the development of effective treatment. Here, using single-cell RNA sequencing, we characterized the peripheral blood mononuclear cells (PBMCs) from uninfected controls and COVID-19 patients and cells in paired broncho-alveolar lavage fluid (BALF). We found a close association of decreased dendritic cells (DCs) and increased monocytes resembling myeloid-derived suppressor cells (MDSCs), which correlated with lymphopenia and inflammation in the blood of severe COVID-19 patients. Those MDSC-like monocytes were immune-paralyzed. In contrast, monocyte-macrophages in BALFs of COVID-19 patients produced massive amounts of cytokines and chemokines, but secreted little interferons. The frequencies of peripheral T cells and NK cells were significantly decreased in severe COVID-19 patients, especially for innate-like T and various CD8+ T cell subsets, compared to healthy controls. In contrast, the proportions of various activated CD4+ T cell subsets among the T cell compartment, including Th1, Th2, and Th17-like cells were increased and more clonally expanded in severe COVID-19 patients. Patients' peripheral T cells showed no sign of exhaustion or augmented cell death, whereas T cells in BALFs produced higher levels of IFNG, TNF, CCL4, CCL5, etc. Paired TCR tracking indicated abundant recruitment of peripheral T cells to the severe patients' lung. Together, this study comprehensively depicts how the immune cell landscape is perturbed in severe COVID-19.

Clinical characteristics of recovered COVID-19 patients with re-detectable positive RNA test.

BACKGROUND: The characteristics, significance and potential cause of positive SARS-CoV-2 diagnoses in recovered coronavirus disease 2019 (COVID-19) patients post discharge (re-detectable positive, RP) remained elusive. METHODS: A total of 262 COVID-19 patients discharged from January 23 to February 25, 2020 were enrolled into this study. RP and non-RP (NRP) patients were grouped according to disease severity, and the characterization at re-admission was analyzed. SARS-CoV-2 RNA and plasma antibody levels were measured, and all patients were followed up for at least 14 days, with a cutoff date of March 10, 2020. RESULTS: A total of 14.5% of RP patients were detected. These patients were characterized as young and displayed mild and moderate conditions compared to NRP patients while no severe patients were RP. RP patients displayed fewer symptoms but similar plasma antibody levels during their hospitalization compared to NRP patients. Upon hospital readmission, these patients showed no obvious symptoms or disease progression. All 21 close contacts of RP patients were tested negative for viral RNA and showed no suspicious symptoms. Eighteen out of 24 of RNA-negative samples detected by the commercial kit were tested positive for viral RNA using a hyper-sensitive method, suggesting that these patients were potential carriers of the virus after recovery from COVID-19. CONCLUSIONS: Our results indicated that young patients, with a mild diagnosis of COVID-19 are more likely to display RP status after discharge. These patients show no obvious symptoms or disease progression upon re-admission. More sensitive RNA detection methods are required to monitor these patients. Our findings provide information and evidence for the management of convalescent COVID-19 patients.

Tocilizumab exerts anti-inflammatory activity in six critically ill COVID-19 patients: a retrospective analysis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has become a worldwide pandemic, affecting countries across the globe. With no current vaccine, treatment is still a critical intervention for minimizing morbidity and preventing disease-specific mortality. This study aimed to assess the clinical outcomes of critically ill COVID-19 patients using Tocilizumab treatment to provide recommendations for the treatment of COVID-19 patients with severe disease. METHODS: This was a retrospective analysis of medical records of six critically ill patients admitted to the Third People's Hospital of Shenzhen, China, from January 11 to February 26, 2020. Patient-related outcomes, including demographic, clinical, and laboratory characteristics before and after the initiation of Tocilizumab, were descriptively analyzed. Four to eight milligrams (mg)/kilogram (kg) of Tocilizumab was prescribed, with Chinese treatment guidelines. RESULTS: By the end of the last follow-up, Patient 1 and Patient 2 developed complications and died after using Tocilizumab for three to four days. Patient 4 died of multiple organ failure caused by cerebral infarction after using Tocilizumab for 39 days. Patient 3 and Patient 6 were discharged after 29 days and 33 days on Tocilizumab, respectively. Clinical symptoms, including fever, heart rate, and oxygen levels, improved after Tocilizumab use. Two patients appeared transient abnormal of liver or renal function indicator, and they can gradually recover. All elevated serum levels of inflammatory factors gradually decreased, except in Patient 2. Patient 3 and Patient 6's inflammatory lesions also significantly improved after initiating Tocilizumab. CONCLUSIONS: Anti-inflammatory treatment with Tocilizumab was found to improve inflammatory responses in critically ill COVID-19 patients. Although some side reactions will occur, patients can gradually recover without affecting the efficacy of the therapy. However, the proper timing to start patients on Tocilizumab patients should be explored. Further prospective, randomized controlled clinical trials are called for.

Single cell RNA sequencing of 13 human tissues identify cell types and receptors of human coronaviruses.

The new coronavirus (SARS-CoV-2) outbreak from December 2019 in Wuhan, Hubei, China, has been declared a global public health emergency. Angiotensin I converting enzyme 2 (ACE2), is the host receptor by SARS-CoV-2 to infect human cells. Although ACE2 is reported to be expressed in lung, liver, stomach, ileum, kidney and colon, its expressing levels are rather low, especially in the lung. SARS-CoV-2 may use co-receptors/auxiliary proteins as ACE2 partner to facilitate the virus entry. To identify the potential candidates, we explored the single cell gene expression atlas including 119 cell types of 13 human tissues and analyzed the single cell co-expression spectrum of 51 reported RNA virus receptors and 400 other membrane proteins. Consistent with other recent reports, we confirmed that ACE2 was mainly expressed in lung AT2, liver cholangiocyte, colon colonocytes, esophagus keratinocytes, ileum ECs, rectum ECs, stomach epithelial cells, and kidney proximal tubules. Intriguingly, we found that the candidate co-receptors, manifesting the most similar expression patterns with ACE2 across 13 human tissues, are all peptidases, including ANPEP, DPP4 and ENPEP. Among them, ANPEP and DPP4 are the known receptors for human CoVs, suggesting ENPEP as another potential receptor for human CoVs. We also conducted "CellPhoneDB" analysis to understand the cell crosstalk between CoV-targets and their surrounding cells across different tissues. We found that macrophages frequently communicate with the CoVs targets through chemokine and phagocytosis signaling, highlighting the importance of tissue macrophages in immune defense and immune pathogenesis.