Epigenetic and transcriptomic reprogramming in monocytes of severe COVID-19 patients reflects alterations in myeloid differentiation and the influence of inflammatory cytokines (preprint)

COVID-19 manifests with a wide spectrum of clinical phenotypes, ranging from asymptomatic and mild to severe and critical. Severe and critical COVID-19 patients are characterized by marked changes in the myeloid compartment, especially monocytes. However, little is known about the epigenetic alterations that occur in these cells during hyperinflammatory responses in severe COVID-19 patients. In this study, we obtained the DNA methylome and transcriptome of peripheral blood monocytes from severe COVID-19 patients. DNA samples extracted from CD14+CD15- monocytes of 48 severe COVID-19 patients and 11 healthy controls were hybridized on MethylationEPIC BeadChip arrays. In parallel, single-cell transcriptomics of 10 severe COVID-19 patients were generated. CellPhoneDB was used to infer changes in the crosstalk between monocytes and other immune cell types. We observed DNA methylation changes in CpG sites associated with interferon-related genes and genes associated with antigen presentation, concordant with gene expression changes. These changes significantly overlapped with those occurring in bacterial sepsis, although specific DNA methylation alterations in genes specific to viral infection were also identified. We also found these alterations to comprise some of the DNA methylation changes occurring during myeloid differentiation and under the influence of inflammatory cytokines. A progression of DNA methylation alterations in relation to the Sequential Organ Failure Assessment (SOFA) score was found to be related to interferon-related genes and T-helper 1 cell cytokine production. CellPhoneDB analysis of the single-cell transcriptomes of other immune cell types suggested the existence of altered crosstalk between monocytes and other cell types like NK cells and regulatory T cells. Our findings show the occurrence of an epigenetic and transcriptional reprogramming of peripheral blood monocytes, which could be associated with the release of aberrant immature monocytes, increased systemic levels of pro-inflammatory cytokines, and changes in immune cell crosstalk in these patients.

Exposing and Overcoming Limitations of Clinical Laboratory Tests in COVID-19 by Adding Immunological Parameters (preprint)

Background: Almost two years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted, nor new tests identified to improve the prediction and management of SARS-CoV-2 infection. Methods: Retrospective observational analysis of the predictive performance of clinical parameters and laboratory tests in hospitalised patients with COVID-19. Outcomes were 28-day survival and maximal severity in a cohort of 1,579 patients and two validation cohorts of 598 and 434 patients. A pilot study conducted in a patient subgroup measured 17 cytokines and 27 lymphocyte phenotypes to explore additional predictive laboratory tests. Findings: 1) Despite a strong association of 22 clinical and laboratory variables with the outcomes, their joint prediction power was limited due to redundancy. 2) Eight variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the statistical predictive power. 3) The interpretation of clinical and laboratory variables was improved by grouping them in categories. 4) Age and organ damage-related tests were the best predictors of survival, and inflammatory-related tests were the best predictors of severity. 5) The pilot study identified several immunological tests (including chemokine ligand 10, chemokine ligand 2, and interleukin 1 receptor antagonist), that performed better than currently used tests. Conclusions: Currently used tests for clinical management of COVID 19 patients are of limited value due to redundancy, as all measure aspects of two major processes: inflammation, and organ damage. There are no independent predictors based on the quality of the nascent adaptive immune response. Understanding the limitations of current tests would improve their interpretation and simplify clinical management protocols. A systematic search for better biomarkers is urgent and feasible.

Recovery of serum testosterone levels is an accurate predictor of survival from COVID-19 in male patients (preprint)

Infection with SARS-CoV-2 portends a broad range of outcomes, from a majority of asymptomatic cases or mild clinical courses to a lethal disease. Robust correlates of severe COVID-19 include old age, male sex, poverty and co-morbidities such as obesity, diabetes or cardiovascular disease. A precise knowledge is still lacking of the molecular and biological mechanisms that may explain the association of severe disease with male sex. Here, we show that testosterone trajectories are highly accurate individual predictors (AUC of ROC = 0.928, p < 0.0001) of survival in male COVID-19 patients. Longitudinal determinations of blood levels of luteinizing hormone (LH) and androstenedione suggest an early modest inhibition of the central LH-androgen biosynthesis axis in a majority of patients, followed by either full recovery in survivors or a peripheral failure in lethal cases. Moreover, failure to reinstate physiological testosterone levels was associated with evidence of impaired T helper differentiation and decrease of non-classical monocytes. The strong association of recovery or failure to reinstate testosterone levels with survival or death from COVID-19 in male patients is suggestive of a significant role of testosterone status in the immune responses to COVID-19.

T-cell responses as a correlate of COVID-19 vaccination. A pilot study in Health Care Workers. (preprint)

Background: Clinical trials on the different vaccines to SARS-CoV-2 have demonstrated protection efficacy, but it is urgent to assess the levels of protection generated with real-world data, especially in individuals professionally exposed. Measuring T-cell responses may complement antibody tests currently in use as correlates of protection but there are not validated T-cell responses applicable to large number of samples. Objective: To assess the feasibility of using T-cell responses to SARS-CoV-2 S peptides by commercially available whole blood interferon-gamma release assays (IGRA) as a correlate of protection. Patients: Twenty health care workers before and after vaccination. Methods: Antibody test to SARS-CoV-2 N and S proteins in parallel with one IGRA assay and two detection techniques than can be automated. Results: IGRA test detected T-cell responses in naturally exposed and vaccinated HCW already after first vaccination dose. the correlation by the two detection methods, CLIA and ELISA, very high (R>0.9) and sensitivity and specificity ranged between 100 and 86% and 100-73% respectively. Even though there was a very high concordance between antibody and the IGRA assay in the ability to detect immune response to SARS-CoV-2 there was a relatively low quantitative correlation. In the small group primed by natural infection, one vaccine dose was sufficient to reach immune response plateau. IGRA was positive in one Ig (S) antibody negative vaccinated immunosuppressed HCW illustrating another advantage of the IGRA test. Conclusion: Whole blood IGRA tests amenable to automation, as the one here reported, constitute a promising additional tool for measuring the state of the immune response to SARS-CoV-2; they are applicable to large number of samples and may become valuable correlates of protection to COVID-19, particularly for vulnerable groups at risk of being re-exposed to infection, as are health care workers.