Prolonged T-cell activation and long COVID symptoms independently associate with severe disease at 3 months in a UK cohort of hospitalized COVID-19 patients

COVID-19 causes immune perturbations which may persist long-term, and patients frequently report ongoing symptoms for months after recovery. We assessed the extent and nature of immune activation at 3 months post hospital admission in patients with mild, moderate or severe COVID-19 and investigated whether immune activation associates with disease severity and long COVID. Patients with severe disease displayed persistent activation of CD4+ and CD8+ T-cells, based on expression of HLA-DR, CD38, Ki67 and granzyme B, but they lacked activation of other immune subsets. Elevated plasma levels of IL-4, IL-7, IL-17 and TNF- were present in patients with severe compared to mild and/or moderate disease. Plasma from severe patients caused T-cells from healthy donors to upregulate IL-15R, suggesting that factors in the plasma of severe patients may increase T-cell responsiveness to IL-15-driven bystander" activation, which may drive persistent T-cell activation after severe COVID-19. Patients with severe disease reported a higher number of long COVID symptoms which correlated with the frequency of two subsets of activated CD4+ and CD8+ T cells (CD4+ T-cell population 2 and CD8+ T-cell population 4; FDR p<0.05), however these associations were lost after adjusting for age, sex and disease severity. Our data suggests that persistent immune activation and long COVID correlate independently with severe disease.

Development and evaluation of low-volume tests to detect and characterise antibodies to SARS-CoV-2

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilised pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID- 19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in- house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterised samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.

Neutrophils in severe COVID-19 are characterized by a hyperactive immature state and maintained CXCR2 expression

Neutrophils are vital in defence against pathogens but excessive neutrophil activity can lead to tissue damage and promote acute respiratory distress syndrome (ARDS). COVID-19 is associated with systemic expansion of immature neutrophils but the functional consequences of this shift to immaturity are not understood. We used flow cytometry to investigate activity and phenotypic diversity of circulating neutrophils in acute and convalescent COVID-19 patients. First, we demonstrate hyperactivation of immature CD10- subpopulations in severe disease, with elevated markers of secondary granule release. Partially activated immature neutrophils were detectable three months post symptom onset, indication long term myeloid dysregulation in convalescent COVID-19 patients. Second, we demonstrate that neutrophils from moderately ill patients downregulate the chemokine receptor CXCR2, while neutrophils from severely ill individuals failed to do so, suggesting altered ability for organ trafficking and a potential mechanism for induction of disease tolerance. CD10-and CXCR2hi neutrophil subpopulations were enriched in severe disease and may represent prognostic biomarkers for identification of individuals at high risk of progressing to severe COVID-19.