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

Introduction: Since December 2019, COVID-19 has spread rapidly across 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 patients after allogeneic haematopoietic stem cell transplantation. Immunological protection against COVID-19 is mediated by both shortlived neutralising 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. Methods: Activation of CD4+ T cells from 18 COVID-19 patients was determined by flow cytometry, both ex vivo and after in vitro restimulation with SARS-CoV-2 Spike and Nucleocapsid antigens. Immunodominant, 15-mer peptides were identified using epitope mapping. T cells clones specific for these epitopes were further chararacterised for the sensitivity and polarisation of their cytokine responses after in vitro restimulation, by ELISA and cytometric assay. Next-generation sequencing revealed fulllength, paired T Cell Receptor (TCR) αβ sequences. Results: We identified three patients with strong CD4+ T cells to SARSCoV- 2 antigens. From these patients, 81 T cell clones specific for a selection of 9 immunodominant epitopes (7 Spike and 2 Nucleocapsid epitopes) were generated. Cytokine analysis showed that the sensitivity and polarisation of T cell responses varied depending on the specific epitope. Moreover, TCRαβ sequences revealed an epitope-dependent difference in the level of clonality. Conclusions: We provide detailed information on SARS-CoV-2-specific CD4+ T cells, including their antigen-specificity, the nature of their cytokine responses and the full sequence of their TCRαβ. These cells have the potential to direct an effective immune response in COVID-19 patients. Our results form a crucial first step towards T cell therapy. Efforts are underway to develop transgenic CD4+ T cells that express the SARS-CoV- 2-specific TCRs identified.

Severity of covid-19 disease is related to hyperactivation and altered migratory capacity of t cells

Introduction: COVID-19 is a life-threatening disease leading to bilateral pneumonia and respiratory failure. The underlying reasons why a smaller percentage of patients present with severe pulmonary symptoms whereas the majority is only mildly affected, are to date not well understood. Methods: We analyzed immune cell dynamics in patients with mild (NIH Score 4+5) and severe (NIH Score 1-3) cases of SARS-CoV2 infection. Patients with mild courses presented with COVID-19 specific, mostly respiratory symptoms, however no one required treatment in the ICU, mechanical ventilation nor died due to COVID-19. Patients with a severe course of disease were treated in the ICU department of our hospital, all of them needed supplementary oxygen. Mortality rate of the cohort with severely ill patients was 33%. Results: Given that T cells play a critical role in the elimination of viral infections, we studied this lymphoid compartment in COVID-19 patients. Compared to healthy controls, patients with SARS-CoV2 infection presented increased T cell activation and proliferation and a decline of naive CD8 T-cells. Interestingly, activation markers on T cells were further enhanced in patients with severe courses of COVID-19 disease. Furthermore, the CD8 T-cell compartment in patients with mild COVID-19 exhibited a shift towards terminal differentiation. In contrast, severe cases showed an expansion of effector cells, which correlated with increased cell activation. To determine whether T-cells might be primed for homing to specific tissues during SARS-CoV-2 infection, we checked for particular chemokine receptors in our cohorts. We found the lung homing receptor CCR4 and the proinflammatory receptor CCR5 strongly upregulated on CD8 T-cells in patients with severe COVID-19 infection, mainly characterized by pulmonary failure and requirement of mechanical ventilation, however not in mild COVID-19 disease. Moreover, CD8 T-cells from patients with severe disease exhibited reduced CCR7 expression, pointing towards enhanced attracting to sites of inflammation and limited homing to secondary lymphoid organs. Further analysis revealed a linear relationship between CCR4 expression and CD8 T-cell activation and effector differentiation in patients with severe disease. Conclusion: Taken together, our data support the critical involvement of T cells in the pathogenesis of SARS-CoV2 infection and link clinical severity to T-cell hyperactivation and altered migratory capacity.

Successful treatment of COVID-19 with convalescent plasma in B-cell depleted patients may promote cellular immunity

Introduction: Treatment with convalescent plasma has been shown to be safe in COVID-19 infection, however, there remain conflicting results regarding its efficacy in immunocompetent patients. Nevertheless, neutralizing antibodies are a key requisite in the fight against viral infections. Patients depleted of antibody producing B-cells such as those treated with rituximab e.g. for hematological malignancies lack a fundamental part of their adaptive immunity. Therefore, treatment with convalescent plasma might still be of benefit in this particularly vulnerable cohort. Methods: Peripheral blood samples were taken from three COVID-19 patients, who were B cell-depleted after previous rituximab treatment, during the course of treatment with convalescent plasma. Flow cytometric analysis of activation markers with or without prior in vitro stimulation with SARS-CoV-2 spike and nucleocapsid antigen revealed antigen-specific T cell responses. Serum levels of SARS-CoV-2-specific IgG and IgM antibodies were measured by automated CLIA. RT-PCR was used to monitor viral load in nasopharyngeal swabs. Results: All three patients made a full recovery from infection following convalescent treatment, even though serum antibody levels decline rapidly after each plasma administration. The recovery coincided with an increase in SARS-CoV-2-specific CD4+ and CD8+ T cells in peripheral blood. Conclusions: We demonstrate efficacy of treatment with convalescent plasma in three patients unable to mount an antibody response by themselves and for the first time show that although application of convalescent plasma only leads to short-lived detectable systemic antibody levels, it appears to boost long-lasting specific T-cell responses.