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Cracking the COVID code: how single cell sequencing changed the way we responded to the pandemic

The ongoing COVID-19 pandemic has been described as “an explosive pandemic of historic proportions” with over 450 million confirmed cases and over 6 million confirmed deaths in less than 3 years worldwide. Our early response to such fast pandemic dynamics included physical prevention of the spreading of the virus and utilizing therapeutic intervention to ameliorate the symptoms and prevent death. It was clear from the beginning that there was an urgent need to understand SARS-CoV-2 interactions with host cells and the host immune response to develop vaccines, improve symptom treatments and understand long term effects of COVID-19 in convalescent patients.

In the past 2 years, more than 250 COVID-19-related studies utilized single cell sequencing, which permanently changed the way we approach modern pandemic dynamics. The technology allowed us to dissect the heterogeneity of immune responses among individual cells and uncover molecular mechanisms of COVID-19 pathogenesis in a very short time. This involved characterizing detailed distribution and transcription profiles of immune cell types involved in the disease, like NK cells [1,2], neutrophils [3], monocytes [4], macrophages [5], and dendritic cells [6], as well as sequencing the T cell and B cell receptor repertoires for vaccine and neutralizing antibody (nAb) treatment development. [7,8] These data can also be used to discover new cell types and subtypes implicated in COVID-19 progression and convalescence, which can be focused as potential new treatment targets. [9,10]

COVID-19 affects many organ systems, either directly by viral infection or by collateral effects of the immune response, like inflammation and cytotoxic tissue damage [11], and these tissue samples can be obtained by biopsy or autopsy for single cell sequencing [13,14]. Also, systemic immune cell interaction profiles can be obtained by single cell sequencing of PBMCs or sorted blood cell populations of interest [12]. Single cell multiomics studies allow for a multi-modal perspective on COVID-19 immune states, such as combining chromatin accessibility or epitope expression with transcriptome sequencing on a single cell level, which allows scientists to study the epigenetic retention of T-cell responses in convalescent patients or during re-infection with a new strain. BCR sequencing of Receptor Binding Domain (RBD)-binding clonotypes from convalescent patients allows for fast discovery of high affinity nAbs. Finally, mining single cell public databases and performing integrative analyses and meta-data analyses provides the means to identify risk and protection correlates against primary and secondary endpoints in vaccine efficacy trials. Here we show how Singleron technology helped in addressing some of these points mentioned above.

Network map of host/viral interaction

The pathophysiology of coronavirus disease 19 (COVID-19) involves a multitude of host responses, yet how they unfold during the course of disease progression remains unclear. Tan et al. performed an integrative analysis of clinical laboratory parameters in blood samples collected from almost a thousand COVID-19 patients and compared the transcriptomes of patient derived BALF and PBMC (monocytes and lymphocytes) to trace the origin of aberrant circulating cytokines, chemokines, and other secreted proteins [12]. Single cell sequencing analysis revealed that low T cell response is associated with overactivation of alveolar macrophages which is correlated with the severity of the disease. Alveolar microenvironment was demonstrated to be the source of most of the cytokine response pathways which releases cytokines into the circulatory system. The pathway enrichment analysis showed that infection phase activates the innate immunity, while convalescence activates the B cell response. In this study, scientists leveraged the power of single cell sequencing to construct a network map of host/viral interaction.

Lung viral-entry gene expression in children and adults

Children have been thought to have higher resilience to SARS-CoV-2 infection, exhibiting less severe symptoms and higher survival rate compared to adults. However, a recent study demonstrated that children and adults have comparable chances of infection by SARS-CoV-2 when exposed to similar environment. It was hypothesized that the expression levels of viral-entry genes might contribute to the milder symptoms observed in children but has not yet been confirmed. To test this, Tao et al. analyzed the expression levels of viral-entry genes (i.e., ACE2, TMPRSS2, and FURIN) in both children and adult lung tissues by single cell RNA sequencing [13]. Both TMPRSS2 and FURIN showed higher expression levels in children compared to adults on protein level and no differences on mRNA level. This was the first study to describe the features of SARS-CoV-2 affected children’s lungs by single cell transcriptome analysis and showed that viral-entry genes might not contribute to the milder symptoms observed in children.

Organotropism of SARS-CoV-2 in autopsy samples

Postmortem examination is a valuable tool for studying the pathobiology of COVID-19. The organotropism of SARS-CoV-2 and the port of virus entry for systemic dissemination remain largely unknown. Yao et al. investigated whether SARS-CoV-2 virus was present in critical immune cell populations like monocytes and macrophages and performed single cell RNA sequencing of lung tissues from an autopsy [14]. They found that the cellular components of alveolar exudate were mainly CD68+ macrophages positive for SARS-CoV-2 spike protein. CD14+ Monocytes and MoAM expressed BSG, TFRC, and NRP1, but not ACE2, suggesting that CD147, transferrin receptor-1, or neuropilin-1 might mediate SARS-CoV-2 infection of monocytes and macrophages. The protein was also present in CD34+ endothelia at blood–air barrier and pulmonary vessels in serial sections of the COVID-19 lungs, raising the possibility that SARS-CoV-2 was able to infiltrate blood–air barrier for intrapulmonary and systemic dissemination. Dissecting infiltrating immune cell transcriptomic profiles of different organ sections, as well as resident tissue cells, provides novel insights into the organotropism of SARS-CoV-2 and mechanisms of viral dissemination.

ACE2 expression in lung and bladder cell populations

Lung is not the only organ affected by COVID-19. The disease was shown to cause acute kidney injury and the virus was also detected in urine. Angiotensin-converting enzyme II (ACE2) has been proposed to serve as the receptor for entry of SARS-CoV-2, which is the same as that for the severe acute respiratory syndrome. Lin et al. investigated the possible causes of kidney damage and the potential route of SARS-CoV-2 infection in the urinary system [15]. They utilized data mining of kidney and bladder single cell atlas and performed kidney single-cell RNA sequencing to evaluate ACE2 gene expression in all cell types. The data revealed that ACE2 expression was detected in proximal convoluted tubule, proximal tubule and proximal straight tubule cells of the kidney, as well as bladder epithelial cells, intermediate cells and umbrella cells. Pathway enrichment analysis also confirmed normal functions of ACE2, such as the renin-angiotensin system, PT bicarbonate reclamation, mineral absorption, and multiple metabolism-related pathways. This study is a good example of how data mining of single cell databases can provide evidence of potential routes of SARS-CoV-2 infection in different organ systems. This allows for a more precise experimental or clinical study design, saving time and lowering costs.

ACE2 expression in liver cholangiocytes

Liver is the second most frequently affected organ by SARS-CoV-2. A substantial proportion of COVID-19 patients showed signs of various degrees of liver damage, for which the mechanism and implication has yet to be determined. Chai et al. used single cell sequencing and data mining to identify cell type specific ACE2 expression in cholangiocytes of the healthy liver from two independent cohorts [16]. The results indicated that the virus might directly bind to ACE2 positive cholangiocytes and not necessarily hepatocytes, implicating the dysfunction of these cells as one of the possible causes of liver abnormalities detected in COVID-19 patients. This study demonstrated the highly sensitive nature of single-cell resolution analysis and facilitated the understanding of the mechanisms of liver malfunction in SARS-CoV-2 infected patients.

Effect of chemotherapy on COVID-19 convalescent cancer patients

Cancer patients usually have a weakened immune system and autoreactive responses and were reported to be more susceptible to SARS-CoV-2 infection, with higher mortality rate compared to regular COVID-19 patients. Previous studies suggested that memory B cells (MBCs) against SARS-CoV-2 could be enriched for up for six months in the general convalescent patients, while levels of anti-SARS-CoV-2 IgG antibody rapidly declined as early as three months after infection. Huang et al. used single cell sequencing to better comprehend the effects of chemotherapy and radiotherapy on the durability of anti-SARS-CoV-2 antibodies and their impact on the immune system of COVID-19 cancer patients [17]. They showed that patients undergoing these treatments have longer SARS-COV-2 IgG retention and exhibit persistent activation of CD8+ T-cells, memory B-cells, and plasma cells. Single cell transcriptomic profiling is a powerful tool for identifying cell subpopulations that are key players in COVID-19 convalescence, especially in patients with comorbidities and undergoing specific treatments.

A Message to the Pandemic: Lines Have Been Drawn!

Rapid development of single cell sequencing technologies was leveraged for a swift response to the pandemic, accelerating vaccine and treatment development, as well as tackling numerous problems arising in convalescent patients as the consequences of the disease. Armed with lessons learned in how we approached the pandemic and the technologies that allow us to define viral entry-markers in specific cell types, profile the immune state of viral infection, map the virus/host interaction and identify key cell type populations in each organ affected, we are well equipped to take better control of this pandemic. Efficient and precise reactions to identify these factors, as well as quick treatment and vaccine development are key to standing our ground against future outbreaks.

[1] Maucourant et al. Natural killer cell immunotypes related to COVID-19 disease severity. Sci. Immunol., 2020. [PubMed]

[2] Witkowski et al. Untimely TGFβ responses in COVID-19 limit antiviral functions of NK cells Nature, 2021. [PubMed]

[3] Barnes et al. Targeting potential drivers of COVID-19: neutrophil extracellular traps. J. Exp. Med., 2020. [PubMed]

[4] Schulte-Schrepping et al. Severe COVID-19 is marked by a dysregulatedmyeloid cell compartment. Cell, 2021. [PubMed]

[5] Merad & Martin. Pathological inflammation in patients with COVID-19: a key role for monocytes and macrophages. Nat. Rev. Immunol, 2020. [PubMed]

[6] Arunachalam et al. Systems biological assessment of immunity to mild versus severe COVID-19 infection in humans. Science, 2020. [PubMed]

[7] Meckiff et al. Imbalance of Regulatory and Cytotoxic SARS-CoV-2-Reactive CD4+ T Cells in COVID-19. Cell, 2020. [PubMed]

[8] Cao et al. Potent neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell sequencing of convalescent patients’ B cells. Cell, 2020. [PubMed]

[9] Gassen et al. SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting antivirals. Nature Commun., 2021. [PubMed]

[10] Wilk et al. A single-cell atlas of the peripheral immune response in patients with severe COVID-19. Nat. Med., 2020. [PubMed]

[11] Georg et al. Complement activation induces excessive T cell cytotoxicity in severe COVID-19. Cell, 2021. [PubMed]

[12] Tan et al. Integrating longitudinal clinical laboratory tests with targeted proteomic and transcriptomic analyses reveal the landscape of host responses in COVID-19. Cell Discovery, 2021. [PubMed]

[13] Tao et al. Preliminary analyses of scRNA sequencing and immunohistochemistry of children’s lung tissues indicate the expression of SARS-CoV-2 entry related genes may not be the key reason for the milder syndromes of COVID-19 in children. Clinical and Translational Medicine, 2021. [PubMed]

[14] Yao et al. A cohort autopsy study defines COVID-19 systemic pathogenesis. Cell Research, 2021. [PubMed]

[15] Lin et al. Single-cell analysis of angiotensin-converting enzyme II expression in human kidneys and bladders reveals a potential route of 2019 novel coronavirus infection. Chinese Medical Journal, 2021. [PubMed]

[16] Chai et al. Specific ACE 2 Expression in Cholangiocytes May Cause Liver Damage After 2019 nCoV Infection. bioRxiv., 2020. [PubMed]

[17] Huang et al. Durable tracking anti‐SARS‐CoV‐2 antibodies in cancer patients recovered from COVID‐19. Scientific Report, 2021. [PubMed]