Artemisinin inhibits neutrophil and macrophage chemotaxis, cytokine production and NET release.

Immune cell chemotaxis to the sites of pathogen invasion is critical for fighting infection, but in life-threatening conditions such as sepsis and Covid-19, excess activation of the innate immune system is thought to cause a damaging invasion of immune cells into tissues and a consequent excessive release of cytokines, chemokines and neutrophil extracellular traps (NETs). In these circumstances, tempering excessive activation of the innate immune system may, paradoxically, promote recovery. Here we identify the antimalarial compound artemisinin as a potent and selective inhibitor of neutrophil and macrophage chemotaxis induced by a range of chemotactic agents. Artemisinin released calcium from intracellular stores in a similar way to thapsigargin, a known inhibitor of the Sarco/Endoplasmic Reticulum Calcium ATPase pump (SERCA), but unlike thapsigargin, artemisinin blocks only the SERCA3 isoform. Inhibition of SERCA3 by artemisinin was irreversible and was inhibited by iron chelation, suggesting iron-catalysed alkylation of a specific cysteine residue in SERCA3 as the mechanism by which artemisinin inhibits neutrophil motility. In murine infection models, artemisinin potently suppressed neutrophil invasion into both peritoneum and lung in vivo and inhibited the release of cytokines/chemokines and NETs. This work suggests that artemisinin may have value as a therapy in conditions such as sepsis and Covid-19 in which over-activation of the innate immune system causes tissue injury that can lead to death.

Nanocurcumin Potently Inhibits SARS-CoV-2 Spike Protein-Induced Cytokine Storm by Deactivation of MAPK/NF-κB Signaling in Epithelial Cells.

Interleukin-mediated deep cytokine storm, an aggressive inflammatory response to SARS-CoV-2 virus infection in COVID-19 patients, is correlated directly with lung injury, multi-organ failure, and poor prognosis of severe COVID-19 patients. Curcumin (CUR), a phenolic antioxidant compound obtained from turmeric (Curcuma longa L.), is well-known for its strong anti-inflammatory activity. However, its in vivo efficacy is constrained due to poor bioavailability. Herein, we report that CUR-encapsulated polysaccharide nanoparticles (CUR-PS-NPs) potently inhibit the release of cytokines, chemokines, and growth factors associated with damage of SARS-CoV-2 spike protein (CoV2-SP)-stimulated liver Huh7.5 and lung A549 epithelial cells. Treatment with CUR-PS-NPs effectively attenuated the interaction of ACE2 and CoV2-SP. The effects of CUR-PS-NPs were linked to reduced NF-κB/MAPK signaling which in turn decreased CoV2-SP-mediated phosphorylation of p38 MAPK, p42/44 MAPK, and p65/NF-κB as well as nuclear p65/NF-κB expression. The findings of the study strongly indicate that organic NPs of CUR can be used to control hyper-inflammatory responses and prevent lung and liver injuries associated with CoV2-SP-mediated cytokine storm.

Phenotypes and Functions of SARS-CoV-2-Reactive T Cells.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which is an ongoing pandemic disease. SARS-CoV-2-specific CD4+ and CD8+ T-cell responses have been detected and characterized not only in COVID-19 patients and convalescents, but also unexposed individuals. Here, we review the phenotypes and functions of SARS-CoV-2-specific T cells in COVID-19 patients and the relationships between SARS-CoV-2-specific T-cell responses and COVID-19 severity. In addition, we describe the phenotypes and functions of SARS-CoV-2-specific memory T cells after recovery from COVID-19 and discuss the presence of SARS-CoV-2-reactive T cells in unexposed individuals and SARS-CoV-2-specific T-cell responses elicited by COVID-19 vaccines. A better understanding of T-cell responses is important for effective control of the current COVID-19 pandemic.

Understanding COVID-19: From Dysregulated Immunity to Vaccination Status Quo.

The development of vaccines against infectious diseases has helped us battle the greatest threat to public health. With the emergence of novel viruses, targeted immunotherapeutics ranging from informed vaccine development to personalized medicine may be the very thing that separates us between life and death. Late in 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), made a remarkable entrance to human civilization, being one of many to cross the species barrier. This review discusses the important aspects of COVID-19, providing a brief overview of our current understanding of dysregulated immune responses developed using various experimental models, a brief outline of experimental models of COVID-19 and more importantly, the rapid development of vaccines against COVID-19.

SARS-CoV-2 Nsp5 Activates NF-κB Pathway by Upregulating SUMOylation of MAVS.

The COVID-19 is an infectious disease caused by SARS-CoV-2 infection. A large number of clinical studies found high-level expression of pro-inflammatory cytokines in patients infected with SARS-CoV-2, which fuels the rapid development of the disease. However, the specific molecular mechanism is still unclear. In this study, we found that SARS-CoV-2 Nsp5 can induce the expression of cytokines IL-1ß, IL-6, TNF-α, and IL-2 in Calu-3 and THP1 cells. Further research found that Nsp5 enhances cytokine expression through activating the NF-κB signaling pathway. Subsequently, we investigated the upstream effectors of the NF-κB signal pathway on Nsp5 overexpression and discovered that Nsp5 increases the protein level of MAVS. Moreover, Nsp5 can promote the SUMOylation of MAVS to increase its stability and lead to increasing levels of MAVS protein, finally triggering activation of NF-κB signaling. The knockdown of MAVS and the inhibitor of SUMOylation treatment can attenuate Nsp5-mediated NF-κB activation and cytokine induction. We identified a novel role of SARS-CoV-2 Nsp5 to enhance cytokine production by activating the NF-κB signaling pathway.

SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Promote a Proinflammatory Activation Profile on Human Dendritic Cells.

Dendritic cells (DCs) are the most potent antigen-presenting cells, and their function is essential to configure adaptative immunity and avoid excessive inflammation. DCs are predicted to play a crucial role in the clinical evolution of the infection by the severe acute respiratory syndrome (SARS) coronavirus (CoV)-2. DCs interaction with the SARS-CoV-2 Spike protein, which mediates cell receptor binding and subsequent fusion of the viral particle with host cell, is a key step to induce effective immunity against this virus and in the S protein-based vaccination protocols. Here we evaluated human DCs in response to SARS-CoV-2 S protein, or to a fragment encompassing the receptor binding domain (RBD) challenge. Both proteins increased the expression of maturation markers, including MHC molecules and costimulatory receptors. DCs interaction with the SARS-CoV-2 S protein promotes activation of key signaling molecules involved in inflammation, including MAPK, AKT, STAT1, and NFκB, which correlates with the expression and secretion of distinctive proinflammatory cytokines. Differences in the expression of ACE2 along the differentiation of human monocytes to mature DCs and inter-donor were found. Our results show that SARS-CoV-2 S protein promotes inflammatory response and provides molecular links between individual variations and the degree of response against this virus.

Longitudinal Analysis of Inflammatory Response to SARS-CoV-2 in the Upper Respiratory Tract Reveals an Association with Viral Load, Independent of Symptoms.

BACKGROUND: SARS-CoV-2 infection leads to high viral loads in the upper respiratory tract that may be determinant in virus dissemination. The extent of intranasal antiviral response in relation to symptoms is unknown. Understanding how local innate responses control virus is key in the development of therapeutic approaches. METHODS: SARS-CoV-2-infected patients were enrolled in an observational study conducted at the Geneva University Hospitals, Switzerland, investigating virological and immunological characteristics. Nasal wash and serum specimens from a subset of patients were collected to measure viral load, IgA specific for the S1 domain of the spike protein, and a cytokine panel at different time points after infection; cytokine levels were analyzed in relation to symptoms. RESULTS: Samples from 13 SARS-CoV-2-infected patients and six controls were analyzed. We found an increase in CXCL10 and IL-6, whose levels remained elevated for up to 3 weeks after symptom onset. SARS-CoV-2 infection also induced CCL2 and GM-CSF, suggesting local recruitment and activation of myeloid cells. Local cytokine levels correlated with viral load but not with serum cytokine levels, nor with specific symptoms, including anosmia. Some patients had S1-specific IgA in the nasal cavity while almost none had IgG. CONCLUSION: The nasal epithelium is an active site of cytokine response against SARS-CoV-2 that can last more than 2 weeks; in this mild COVID-19 cohort, anosmia was not associated with increases in any locally produced cytokines.

Cytokine Response to SARS-CoV-2 Infection in Children.

The causal connection between serum biomarkers and COVID-19 severity or pathogenicity in children is unclear. The aim of this study was to describe clinical and immunological features of children affected by COVID-19. The secondary aim was to evaluate whether these cytokines could predict severity of COVID-19. All children (aged 0-18) admitted to the Pediatric Emergency Department and tested with nasopharyngeal swab for SARS-CoV-2 were recruited and assigned to three groups: COVID-19, other infections, control group. Clinical and laboratory data of these patients, including circulating cytokine levels, were analyzed in three groups. Fever was the most frequent symptom in COVID-19 (67.3%). Neutropenia was found in the COVID-19 group (p < 0.05); no difference was observed for lymphocyte counts in the three groups. Higher levels of IL-6 and TNF-alpha were found in the COVID-19 group compared to other infections and control groups (p = 0.014 and p = 0.001, respectively). Whereas, in the COVID-19 group, no difference was observed as for the same cytokines among sub-groups of different disease severity (p = 0.7 and p = 0.8). Serum levels of IL-6 and TNF-alpha were higher in COVID-19 children than in children with other infectious diseases, but those levels did not correlate with disease severity. Clinical studies in a large pediatric population are necessary to better define the role of the immune-mediated response in SARS-CoV-2 infections in children.

Characterization of the SARS-CoV-2 Host Response in Primary Human Airway Epithelial Cells from Aged Individuals.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), a global pandemic characterized by an exaggerated immune response and respiratory illness. Age (>60 years) is a significant risk factor for developing severe COVID-19. To better understand the host response of the aged airway epithelium to SARS-CoV-2 infection, we performed an in vitro study using primary human bronchial epithelial cells from donors >67 years of age differentiated on an air-liquid interface culture. We demonstrate that SARS-CoV-2 infection leads to early induction of a proinflammatory response and a delayed interferon response. In addition, we observed changes in the genes and pathways associated with cell death and senescence throughout infection. In summary, our study provides new and important insights into the temporal kinetics of the airway epithelial innate immune response to SARS-CoV-2 in older individuals.

Live attenuated coronavirus vaccines deficient in N7-Methyltransferase activity induce both humoral and cellular immune responses in mice.

Coronaviruses (CoVs) can infect a variety of hosts, including humans, livestock and companion animals, and pose a serious threat to human health and the economy. The current COVID-19 pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed millions of people. Unfortunately, effective treatments for CoVs infection are still lacking, suggesting the importance of coronavirus vaccines. Our previous work showed that CoV nonstuctural protein 14 (nsp14) functions as (guanine-N7)-methyltransferase (N7-MTase), which is involved in RNA cap formation. Moreover, we found that N7-MTase is well conserved among different CoVs and is a universal target for developing antivirals against CoVs. Here, we show that N7-MTase of CoVs can be an ideal target for designing live attenuated vaccines. Using murine hepatitis virus strain A59 (MHV-A59), a representative and well-studied model of coronaviruses, we constructed N7-MTase-deficient recombinant MHV D330A and Y414A. These two mutants are highly attenuated in mice and exhibit similar replication efficiency to the wild-type (WT) virus in the cell culture. Furthermore, a single dose immunization of D330A or Y414A can induce long-term humoral immune responses and robust CD4+ and CD8+ T cell responses, which can provide full protection against the challenge of a lethal-dose of MHV-A59. Collectively, this study provides an ideal strategy to design live attenuated vaccines for coronavirus by abolishing viral RNA N7-MTase activity. This approach may apply to other RNA viruses that encode their own conservative viral N7-methyltransferase.

Systems Immunology Analysis Reveals the Contribution of Pulmonary and Extrapulmonary Tissues to the Immunopathogenesis of Severe COVID-19 Patients.

As one of the current global health conundrums, COVID-19 pandemic caused a dramatic increase of cases exceeding 79 million and 1.7 million deaths worldwide. Severe presentation of COVID-19 is characterized by cytokine storm and chronic inflammation resulting in multi-organ dysfunction. Currently, it is unclear whether extrapulmonary tissues contribute to the cytokine storm mediated-disease exacerbation. In this study, we applied systems immunology analysis to investigate the immunomodulatory effects of SARS-CoV-2 infection in lung, liver, kidney, and heart tissues and the potential contribution of these tissues to cytokines production. Notably, genes associated with neutrophil-mediated immune response (e.g. CXCL1) were particularly upregulated in lung, whereas genes associated with eosinophil-mediated immune response (e.g. CCL11) were particularly upregulated in heart tissue. In contrast, immune responses mediated by monocytes, dendritic cells, T-cells and B-cells were almost similarly dysregulated in all tissue types. Focused analysis of 14 cytokines classically upregulated in COVID-19 patients revealed that only some of these cytokines are dysregulated in lung tissue, whereas the other cytokines are upregulated in extrapulmonary tissues (e.g. IL6 and IL2RA). Investigations of potential mechanisms by which SARS-CoV-2 modulates the immune response and cytokine production revealed a marked dysregulation of NF-κB signaling particularly CBM complex and the NF-κB inhibitor BCL3. Moreover, overexpression of mucin family genes (e.g. MUC3A, MUC4, MUC5B, MUC16, and MUC17) and HSP90AB1 suggest that the exacerbated inflammation activated pulmonary and extrapulmonary tissues remodeling. In addition, we identified multiple sets of immune response associated genes upregulated in a tissue-specific manner (DCLRE1C, CHI3L1, and PARP14 in lung; APOA4, NFASC, WIPF3, and CD34 in liver; LILRA5, ISG20, S100A12, and HLX in kidney; and ASS1 and PTPN1 in heart). Altogether, these findings suggest that the cytokines storm triggered by SARS-CoV-2 infection is potentially the result of dysregulated cytokine production by inflamed pulmonary and extrapulmonary (e.g. liver, kidney, and heart) tissues.

Distinctive features of severe SARS-CoV-2 pneumonia.

The coronavirus disease 2019 (COVID-19) pandemic is among the most important public health crises of our generation. Despite the promise of prevention offered by effective vaccines, patients with severe COVID-19 will continue to populate hospitals and intensive care units for the foreseeable future. The most common clinical presentation of severe COVID-19 is hypoxemia and respiratory failure, typical of the acute respiratory distress syndrome (ARDS). Whether the clinical features and pathobiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia differ from those of pneumonia secondary to other pathogens is unclear. This uncertainty has created variability in the application of historically proven therapies for ARDS to patients with COVID-19. We review the available literature and find many similarities between patients with ARDS from pneumonia attributable to SARS-CoV-2 versus other respiratory pathogens. A notable exception is the long duration of illness among patients with COVID-19, which could result from its unique pathobiology. Available data support the use of care pathways and therapies proven effective for patients with ARDS, while pointing to unique features that might be therapeutically targeted for patients with severe SARS-CoV-2 pneumonia.

The zinc finger transcription factor, KLF2, protects against COVID-19 associated endothelial dysfunction.

Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its patho-mechanism being incompletely understood. Emerging evidence has demonstrated that endothelial dysfunction precipitates COVID-19 and its accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. The objective of the present study is to evaluate whether kruppel-like factor 2 (KLF2), a master regulator of vascular homeostasis, represents a therapeutic target for COVID-19-induced endothelial dysfunction. Here, we demonstrate that the expression of KLF2 was reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1ß and TNF-α, two cytokines elevated in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Pharmacologic (atorvastatin and tannic acid) and genetic (adenoviral overexpression) approaches to augment KLF2 levels attenuated COVID-19-serum-induced increase in endothelial inflammation and monocyte adhesion. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis, and reduced angiogenesis). Finally, knockdown of KLF2 partially reversed the ameliorative effect of atorvastatin on COVID-19-serum-induced endothelial inflammation and monocyte adhesion. Collectively, the present study implicates loss of KLF2 as an important molecular event in the development of COVID-19-induced vascular disease and suggests that efforts to augment KLF2 levels may be therapeutically beneficial.

The single-cell epigenomic and transcriptional landscape of immunity to influenza vaccination.

Emerging evidence indicates a fundamental role for the epigenome in immunity. Here, we mapped the epigenomic and transcriptional landscape of immunity to influenza vaccination in humans at the single-cell level. Vaccination against seasonal influenza induced persistently diminished H3K27ac in monocytes and myeloid dendritic cells (mDCs), which was associated with impaired cytokine responses to Toll-like receptor stimulation. Single-cell ATAC-seq analysis revealed an epigenomically distinct subcluster of monocytes with reduced chromatin accessibility at AP-1-targeted loci after vaccination. Similar effects were observed in response to vaccination with the AS03-adjuvanted H5N1 pandemic influenza vaccine. However, this vaccine also stimulated persistently increased chromatin accessibility at interferon response factor (IRF) loci in monocytes and mDCs. This was associated with elevated expression of antiviral genes and heightened resistance to the unrelated Zika and Dengue viruses. These results demonstrate that vaccination stimulates persistent epigenomic remodeling of the innate immune system and reveal AS03's potential as an epigenetic adjuvant.

Chloroquine attenuates thymic stromal lymphopoietin production via suppressing caspase-1 signaling in mast cells.

Thymic stromal lymphopoietin (TSLP) produced by mast cells is involved in allergic inflammation pathogenesis. Chloroquine (CQ) is known to be an anti-malarial drug; however, additional protective functions of CQ have been discovered. This study aims to clarify an anti-inflammatory effect of CQ through modulating TSLP levels using an in vitro model of phorbol myristate acetate (PMA) + A23187-activated human mast cell line (HMC-1) and an in vivo model of PMA-irritated ear edema. CQ treatment reduced the production and mRNA expression levels of TSLP in activated HMC-1 cells. CQ down-regulated caspase-1 (CASP1), MAPKs, and NF-κB levels enhanced by stimulation with PMA + A23187. Moreover, ear thickness in ear edema was suppressed following CQ treatment. CQ decreased CASP1 and NF-κB levels in the ear tissue. TSLP levels in the ear tissue and serum were reduced following CQ treatment. Collectively, the above findings elucidate that CQ inhibits the pro-inflammatory mechanisms of TSLP via the down-regulation of distinct intracellular signaling cascade in mast cells. Therefore, CQ may have protective roles against TSLP-mediated inflammatory disorders.

How could perfluorocarbon affect cytokine storm and angiogenesis in coronavirus disease 2019 (COVID-19): role of hypoxia-inducible factor 1α.

Coronavirus disease 2019 (COVID-19) pandemic is still a world-class challenge. Inflammation, especially its severe form with excess release of pro-inflammatory cytokines (cytokine storm) which is a life-threatening condition, is among the most important suspects involved in COVID-19 pathogenesis. It has been shown that cytokine storm could cause notable morbidities such as acute respiratory distress syndrome (ARDS) which leads to hypoxia which is significantly associated with mortality of patients with COVID-19. Hypoxia-inducible factor 1α (HIF-1α) which activates following ARDS-induced hypoxia plays a crucial role in pathogenesis of cytokine storm. The expression of tumor necrosis factor α (TNF-α), interleukin 1 ß (IL-1ß), and IL-6 which are key elements of cytokine storm are by nuclear factor κß (NFκB). Interestingly, during the hypoxia, HIF-1α activates NFκB to induce expression of pro-angiogenic and pro-inflammatory factors. These released factors starts a autocrine/paracrine loop and causes deterioration of their etiological pathways of expression: cytokine storm and ARDS. To sum up, it seems HIF-1α is an important target to hit to ameliorate the mentioned pathways. Herein, we suggest perfluorocarbons (PFCs) which are among the organofluorine compounds as a possible co-treatment to reduce hypoxemia and then hypoxia. These substances are known for their high gas solving potential that make them able to be used as a synthetic artificial blood product. Due to the potential of PFCs to affect the fountain of important physiopathological pathway such as inflammation a hypoxia through affecting NFκB, they could be considered as multi-target co-treatment for ARD individuals with COVID-19. It is highly suggested to evaluate this hypothesis in following researches.

Cytokine Overproduction and Immune System Dysregulation in alloHSCT and COVID-19 Patients.

The COVID-19 pathomechanism depends on (i) the pathogenicity of the virus, (ii) ability of the immune system to respond to the cytopathic effect of the virus infection, (iii) co-morbidities. Inflammatory cytokine production constitutes a hallmark of COVID-19 that is facilitated by inability of adaptive immunity to control virus invasion. The effect of cytokine release syndrome is deleterious, but the severity of it depends on other confounding factors: age and comorbidities. In this study, we analyze the literature data on the post-transplant course of allogeneic hematopoietic stem cell transplanted (alloHSCT) patients, which is affected by generated inflammatory cytokines. The sequence of events boosting cytokine production was analyzed in relation to clinical and laboratory data highlighting the impact of cytokine generation on the post-transplant course. The collected data were compared to those from studies on COVID-19 patients. The similarities are: (i) the damage/pathogen-associated molecular pattern (DAMP/PAMP) stage is similar except for the initiation hit being sterile in alloHSCT (toxic damage of conditioning regimen) and viral in COVID-19; (ii) genetic host-derived factors play a role; (iii) adaptive immunity fails, DAMP signal(s) increases, over-production of cytokines occurs; (iv) monocytes lacking HLADR expression emerge, being suppressor cells hampering adaptive immunity; (v) immune system homeostasis is broken, the patient's status deteriorates to bed dependency, leading to hypo-oxygenation and malnutrition, which in turn stimulates the intracellular alert pathways with vigorous transcription of cytokine genes. All starts with the interaction between DAMPs with appropriate receptors, which leads to the production of pro-inflammatory cytokines, the inflammatory process spreads, tissue is damaged, DAMPs are released and a vicious cycle occurs. Attempts to modify intracellular signaling pathways in patients with post-alloHSCT graft vs host disease have already been undertaken. The similarities documented in this study show that this approach may also be used in COVID-19 patients for tuning signal transduction processes to interrupt the cycle that powers the cytokine overproduction.

Molecular Perspectives of SARS-CoV-2: Pathology, Immune Evasion, and Therapeutic Interventions.

The outbreak of coronavirus disease 2019 (COVID-19) has not only affected human health but also diverted the focus of research and derailed the world economy over the past year. Recently, vaccination against COVID-19 has begun, but further studies on effective therapeutic agents are still needed. The severity of COVID-19 is attributable to several factors such as the dysfunctional host immune response manifested by uncontrolled viral replication, type I interferon suppression, and release of impaired cytokines by the infected resident and recruited cells. Due to the evolving pathophysiology and direct involvement of the host immune system in COVID-19, the use of immune-modulating drugs is still challenging. For the use of immune-modulating drugs in severe COVID-19, it is important to balance the fight between the aggravated immune system and suppression of immune defense against the virus that causes secondary infection. In addition, the interplaying events that occur during virus-host interactions, such as activation of the host immune system, immune evasion mechanism of the virus, and manifestation of different stages of COVID-19, are disjunctive and require thorough streamlining. This review provides an update on the immunotherapeutic interventions implemented to combat COVID-19 along with the understanding of molecular aspects of the immune evasion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which may provide opportunities to develop more effective and promising therapeutics.

Human CD4+ T cells specific for dominant epitopes of SARS-CoV-2 Spike and Nucleocapsid proteins with therapeutic potential.

Since December 2019, Coronavirus disease-19 (COVID-19) has spread rapidly throughout the world, leading to a global effort to develop vaccines and treatments. Despite extensive progress, there remains a need for treatments to bolster the immune responses in infected immunocompromised individuals, such as cancer patients who recently underwent a haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both short-lived neutralizing antibodies and long-lasting virus-reactive T cells. Therefore, we propose that T cell therapy may augment efficacy of current treatments. For the greatest efficacy with minimal adverse effects, it is important that any cellular therapy is designed to be as specific and directed as possible. Here, we identify T cells from COVID-19 patients with a potentially protective response to two major antigens of the SARS-CoV-2 virus, Spike and Nucleocapsid protein. By generating clones of highly virus-reactive CD4+ T cells, we were able to confirm a set of nine immunodominant epitopes and characterize T cell responses against these. Accordingly, the sensitivity of T cell clones for their specific epitope, as well as the extent and focus of their cytokine response was examined. Moreover, using an advanced T cell receptor (TCR) sequencing approach, we determined the paired TCR-αß sequences of clones of interest. While these data on a limited population require further expansion for universal application, the results presented here form a crucial first step towards TCR-transgenic CD4+ T cell therapy of COVID-19.