Evidence of Autoinflammation as the Principal Mechanism of Myocardial Injury in SARS-CoV-2 PCR-Positive Autopsy Hearts (preprint)

Background Disease from Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) remains the third leading cause of death in the United States, after cancer and heart disease. Many patients infected with this virus develop cardiovascular complications including myocardial infarctions, stroke, arrhythmia, heart failure, and sudden cardiac death. Specifically, patients with SARS-CoV-2 have a high prevalence of severe myocardial injury (20–28%). The purpose of this study is to understand the primary mechanism of myocyte injury in patients infected with SARS-CoV-2.Methods We investigated a consecutive cohort of 84 medical examiner cases who died with PCR-positive SARS-CoV-2 (COVpos) infection prior to availability of therapy or vaccines. We compared them to a consecutive cohort of 42 age- and sex-matched controls who were PCR-negative for SARS-CoV-2 (COVneg). Formalin-fixed paraffin embedded sections of left and right ventricle were examined on each case using antibodies directed against CD42 (platelets), CD15 (myeloid cells), CD68 (monocytes), C4d, Fibrin, CD34 (stem cell antigen), CD56 (natural killer cells), and Myeloperoxidase (MPO) (neutrophils and NETs). Slides were scanned using an Aperio slide scanner and viewer and each digital slide was entirely examined at 5x,10x and 20x. Each slide was graded using a 0–3 scale where 3 indicates the marker was present in every field at 20x. We used a Welch 2-sample T-test to determine significance.Results We found a significant difference between COVpos and COVneg samples for all markers, all of which were significant at p < 0.001. The most prominent features were neutrophils (CD15, MPO) and MPO positive debris suggestive of NETS and were located in or around arterioles, venules, and capillaries. A similar distribution of platelets, monocytes, and C4d was seen in COVpos cases. Fibrin was found scattered in arterioles, venules, interstitial regions, and within ventricular cavities. CD34 highlighted vascular alterations of endothelial cells in some but not all cases.Conclusion Autoinflammation is the primary mechanism of myocyte injury observed in COVpos hearts. The significant increase in platelets, monocytes, and neutrophils and the presence of neutrophil NETs, C4d, and fibrin suggest that myocardial injury involves neutrophils, NETosis, coagulation, complement activation, and monocyte accumulation.

Resolving the developmental mechanisms of coagulation abnormalities characteristic of SARS-CoV2 based on single-cell transcriptome analysis (preprint)

The COVID-19 outbreak caused by the SARS-CoV-2 virus has developed into a global health emergency. In addition to causing respiratory symptoms following SARS-CoV-2 infection, COVID-19-associated coagulopathy (CAC) is the main cause of death in patients with severe COVID-19. In this study, we performed single-cell sequencing analysis of the right ventricular free wall tissue from healthy donors, patients who died in the hypercoagulable phase of CAC, and patients in the fibrinolytic phase of CAC. Among these, we collected 61,187 cells, which were enriched in 24 immune cell subsets and 13 cardiac-resident cell subsets. We found that in response to SARS-CoV-2 infection, CD9highCCR2high monocyte-derived mo promoted hyperactivation of the immune system and initiated the extrinsic coagulation pathway by activating CXCR-GNB/G-PI3K-AKT. This sequence of events is the main process contributing the development of coagulation disorders subsequent to SARS-CoV-2 infection. In the characteristic coagulation disorder caused by SARS-CoV-2, excessive immune activation is accompanied by an increase in cellular iron content, which in turn promotes oxidative stress and intensifies intercellular competition. This induces cells to alter their metabolic environment, resulting in an increase in sugar uptake, such as that via the glycosaminoglycan synthesis pathway, in CAC coagulation disorders. In addition, high levels of reactive oxygen species generated in response elevated iron levels promote the activation of unsaturated fatty acid metabolic pathways in endothelial cell subgroups, including vascular endothelial cells. This in turn promotes the excessive production of the toxic peroxidation by-product malondialdehyde, which exacerbates both the damage caused to endothelial cells and coagulation disorders.

Persistently high platelet factor 4 levels in an adolescent with recurrent late thrombotic complications after SARS-CoV-2 mRNA vaccination: A case report (preprint)

Background Thrombosis after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination is a serious complication for patients with a thrombophilic predisposition. Factors that predict the risk of post-vaccination thrombosis should be explored. We report a case in which a patient with pediatric antiphospholipid syndrome (APS) developed deep vein thrombosis (DVT) six months after receiving a second dose of the BNT162b2 vaccine.Case presentation: A 17-year-old girl with no family history of thrombophilia developed DVT at six years of age. The thrombus was found in the right common iliac vein and the inferior vena cava, with concomitant left pulmonary infarction. After treatment with warfarin, the pulmonary infarction was resolved, but the thrombus became organized and persisted for the next 11 years. The patient was treated with anticoagulants for six years after DVT onset, with subsequent cessation of treatment for five years without thrombosis recurrence. She received the BNT162b2 vaccine at 17 years of age, one week before a routine outpatient visit. Elevation of platelet factor 4 level was detected 14 days after the first vaccination and remained for five months after that, but without thrombotic symptoms. A second dose of the BNT162b2 vaccine was administered; six months later, the DVT in the right common iliac vein recurred and was treated with a direct oral anticoagulant.Conclusions The BNT162b2 vaccine exacerbated her antiphospholipid antibody syndrome by activating the coagulation system, thereby exacerbating her thrombosis. Platelet factor 4 may be a useful indicator of the coagulation system. The persistence of high platelet factor 4 levels after vaccination suggests that the vaccine caused DVT by exacerbating the patient’s APS. After vaccination of patients with a predisposition to thrombosis, coagulation status and platelet activation markers should be monitored to prevent the development of DVT.

Sex difference in the cytokine profile among patients hospitalized for COVID-19 and during their recovery: predominance of females in adhesion molecules and males in oxidative stress (preprint)

Background Male sex is associated with greater severity and mortality from coronavirus disease 2019 (COVID-19), although infections are equally distributed between men and women. The present study aims to investigate sex differences in the hyperinflammatory immune response to SARS-CoV-2 infection and consequent thrombosis using linked cytokine profile and blood laboratory data.Methods This observational cohort study involved 99 COVID-19 patients (69 male and 30 female adults), hospitalized between March 2021 and April 2022. Their clinical and laboratory data were collected to examine sex difference in oxidative stress, neutrophil extracellular traps (NETs) formation and plasma cytokines at hospital admission and up to 5 months of their recovery.Results Dihydrotestosterone (DHT) levels were transiently reduced, while sex hormone binding globulin levels were continuously decreased in male post-COVID-19 patients after its rise at diagnosis. Regarding inflammatory cytokines, interleukin-6 (IL-6) and interferon-gamma are generally increased at diagnosis, while IL-6 is decreasing in post-COVID-19 patients. Tumor necrosis factor-alpha had 5-fold increase in females at diagnosis. Chemokines IL-8 and monocyte chemoattractant protein-1 and coagulation markers intercellular adhesion molecule-1 and E-selectin were consistently upregulated in female COVID-19 and post-COVID-19 patients, in contrast to vascular cell adhesion molecule-1 and P-selectin. DHT increased reactive oxygen species in neutrophils of male patients, while estrogen decreased in females. Markers for NETs formation, such as circulating DNA and myeloperoxidase, were significantly increased in the plasma of patients. Sex hormones are mostly negatively correlated with leukocytes, while in positive correlation with coagulation markers.Conclusions Markers of chemotaxis, endothelial dysfunction and inflammation are still detectable and partially sex dependent in COVID-19 patients after 5 months of hospital admission. These distributions of sex steroid hormones splitted endothelial adhesion molecules, coagulation and inflammation factors, while they generalized chemokines.

Nirmatrelvir and the Risk of Post-Acute Sequelae of COVID-19 (preprint)

Long Covid -- the disease encompassing the post-acute sequelae of SARS-CoV-2 (PASC) -- affects millions of people around the world. Prevention of PASC is an urgent public health priority. In this work, we aimed to examine whether treatment with nirmatrelvir in the acute phase of COVID-19 is associated with reduced risk of post-acute sequelae. We used the healthcare databases of the US Department of Veterans Affairs to identify users of the health system who had a SARS-CoV-2 positive test between March 01, 2022 and June 30, 2022, were not hospitalized on the day of the positive test, had at least 1 risk factor for progression to severe COVID-19 illness and survived the first 30 days after SARS-CoV-2 diagnosis. We identify those who were treated with oral nirmatrelvir within 5 days after the positive test (n=9217) and those who received no COVID-19 antiviral or antibody treatment during the acute phase of SARS-CoV-2 infection (control group, n= 47,123). Inverse probability weighted survival models were used to estimate the effect of nirmatrelvir (versus control) on a prespecified panel of 12 post-acute COVID-19 outcomes and reported as hazard ratio (HR) and absolute risk reduction (ARR) in percentage at 90 days. Compared to the control group, treatment with nirmatrelvir was associated with reduced risk of PASC (HR 0.74 95% CI (0.69, 0.81), ARR 2.32 (1.73, 2.91)) including reduced risk of 10 of 12 post-acute sequelae in the cardiovascular system (dysrhythmia and ischemic heart disease), coagulation and hematologic disorders (deep vein thrombosis, and pulmonary embolism), fatigue, liver disease, acute kidney disease, muscle pain, neurocognitive impairment, and shortness of breath. Nirmatrelvir was also associated with reduced risk of post-acute death (HR 0.52 (0.35, 0.77), ARR 0.28 (0.14, 0.41)), and post-acute hospitalization (HR 0.70 (0.61, 0.80), ARR 1.09 (0.72, 1.46)). Nirmatrelvir was associated with reduced risk of PASC in people who were unvaccinated, vaccinated, and boosted, and in people with primary SARS-CoV-2 infection and reinfection. In sum, our results show that in people with SARS-CoV-2 infection who had at least 1 risk factor for progression to severe COVID-19 illness, treatment with nirmatrelvir within 5 days of a positive SARS-CoV-2 test was associated with reduced risk of PASC regardless of vaccination status and history of prior infection. The totality of findings suggests that treatment with nirmatrelvir during the acute phase of COVID-19 reduces the risk of post-acute adverse health outcomes.

Point-of-care Diagnosis and Monitoring of Hypofibrinolysis in the Critically Ill: Results from a Feasibility Study. (preprint)

Background In critical conditions such as sepsis, severe trauma, COVID-19 and non-COVID acute respiratory failure, hypofibrinolysis is associated with multi-organ dysfunction syndrome and death. The mechanisms underpinning hypofibrinolysis may include reduced tissue plasminogen activator (t-PA) and/or plasmin effect due to elevated inhibitor levels, reduced expression and/or exhaustion. This study in critically ill patients with hypofibrinolysis aimed to evaluate the ability of t-PA and plasminogen supplementation to restore fibrinolysis assessed by bedside viscoelastic testing (VET).Methods Prospective observational and interventional studies were undertaken in 28 critically ill patients identified as hypercoagulant and hypofibrinolytic using standard ClotPro VET. Hypercoagulation was defined as above normal values for clot amplitude on the EX-test (tissue factor (TF) activated coagulation) or FIB-test (TF activated coagulation with platelet inhibition). Hypofibrinolysis was defined as a clot lysis time > 300 seconds on the TPA-test (TF activated coagulation with t-PA accelerated fibrinolysis). In experimental VET, repeat TPA-tests were spiked with additional t-PA and/or plasminogen and the effect on lysis time determined. In a hypofibrinolytic patient, alteplase was administered intravenously over a 24-hr period with standard ClotPro VET repeated frequently throughout to monitor the effect on coagulation and fibrinolysis.Results In the ex-vivo studies, distinct response groups emerged with increased fibrinolysis observed following (i) additional t-PA supplementation only, or (ii) combined plasminogen and t-PA supplementation. A baseline TPA-test lysis time of > 1000 sec associated with the latter group. In the interventional study, alteplase administered as a 2-hr bolus (25 mg) followed by a 22-hr infusion (1 mg/hr) resulted in a gradual reduction in serial TPA-test lysis times.Conclusions ClotPro viscoelastic testing, the associated TPA-test and the novel spiked ex-vivo assays may be utilised to (i) investigate the potential mechanisms of hypofibrinolysis, (ii) guide corrective treatment, and (iii) monitor in real-time the treatment effect. Such a precision-medicine and personalised treatment approach to the management of hypofibrinolysis has the potential to increase treatment benefit, whilst minimising adverse events in hypofibrinolytic critically ill patients.Trial Registration: VETtiPAT ARF, a clinical trial evaluating the use of ClotPro-guided tissue plasminogen activator (alteplase) administration in hypofibrinolytic patients with acute respiratory failure is ongoing (ClinicalTrials.gov NCT05540834, registered 15 September 2022, retrospectively registered).

Treatment with anti-inflammatory viral serpin modulates immuno-thrombotic responses and improves outcomes in SARS-CoV-2 infected mice (preprint)

Severe acute respiratory distress syndrome (ARDS) during SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) infection, manifests as uncontrolled lung inflammation and systemic thrombosis with high mortality. Anti-viral drugs and monoclonal antibodies can reduce COVID-19 severity if administered in the early viremic phase, but treatments for later stage immuno-thrombotic syndrome and long COVID are limited. Serine protease inhibitors (SERPINS) regulate activated proteases during thrombotic, thrombolytic and immune responses. The myxoma poxvirus-derived Serp-1 protein is a secreted immunomodulatory serpin that targets activated coagulation and complement protease pathways as part of a self-defense strategy to combat viral clearance by the innate immune system. When purified and utilized as an anti-immune therapeutic, Serp-1 is effective as an anti-inflammatory drug in multiple animal models of inflammatory lung disease and vasculitis. Here, we describe systemic treatment with purified PEGylated Serp-1 (PEGSerp-1) as a therapy for immuno-thrombotic complications during ARDS. Treatment with PEGSerp-1 in two distinct mouse-adapted SARS-CoV-2 models in C57Bl/6 and BALB/c mice reduced lung and heart inflammation, with improved clinical outcomes. PEGSerp-1 significantly reduced M1 macrophage invasion in the lung and heart by modifying urokinase-type plasminogen activator receptor (uPAR) and complement membrane attack complex (MAC). Sequential changes in urokinase-type plasminogen activator receptor (uPAR) and serpin gene expression were observed in lung and heart with PEGSerp-1 treatment. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for treatment of severe viral ARDS with additional potential to reduce late SARS-CoV-2 complications related to immune-thrombotic events that persist during long COVID. Significance: Severe acute respiratory distress syndrome (ARDS) in SARS-CoV-2 infection manifests as uncontrolled tissue inflammation and systemic thrombosis with high mortality. Anti-viral drugs and monoclonal antibodies reduce COVID-19 severity if administered early, but treatments for later stage immuno-thrombosis are limited. Serine protease inhibitors (SERPINS) regulate thrombotic, thrombolytic and complement pathways. We investigate here systemic treatment with purified poxvirus-derived PEGSerp-1 as a therapeutic for immuno-thrombotic complications in viral ARDS. PEGSerp-1 treatment in two mouse-adapted SARS-CoV-2 models (C57Bl/6 and BALB/c) significantly reduced lung and heart inflammation and improved clinical outcomes, with sequential changes in thrombolytic (uPAR) and complement expression. PEGSerp-1 is a highly effective immune-modulator with therapeutic potential for immune-thrombotic complications in severe viral ARDS and has potential benefit for long COVID.

Congenital Bleeding Disorders and COVID-19 - An Emphasis on the Role of Thrombosis as One of the Main Causes of Morbidity and Mortality in COVID-19.

A turbulent coagulation system is a prominent feature of Coronavirus Disease 2019 (COVID-19), with venous thromboembolism (VTE) a leading cause of death. Our hypothesis is that patients with inherited hypocoagulability, like congenital bleeding disorders (CBD), enjoy a protective effect against COVID-19-induced hypercoagulability and related fatal consequences. Our primary and follow-up observations revealed this effect, at least among patients with moderate to severe congenital bleeding disorders, particularly coagulation factor deficiencies. Theoretically, patients with inherited hypocoagulobility have only a potential protective effect against COVID-19-related hypercoagulability. Yet the lower rate of morbidity and mortality in patients with CBDs suggests that hypercoagulability and thrombotic events are the main cause of death in COVID-19. Therefore, appropriate and timely administration of anticoagulants could significantly decrease the rate of morbidity and mortality in COVID-19.

Disentangling the common genetic architecture and causality of rheumatoid arthritis and systemic lupus erythematosus with COVID-19 outcomes: genome-wide cross trait analysis and bi-directional Mendelian randomization study (preprint)

COVID-19 may cause a dysregulation of the immune system and has complex relationships with multiple autoimmune diseases, including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). However, little is known about their common genetic architecture. We analysed summary-level genetic data from the latest COVID-19 host genetics consortium and consortia on RA and SLE to examine the shared genetic etiology and causal relationship between COVID-19 and RA/SLE. The cross-trait meta-analysis identified 46, 47, and 19 shared genetic loci for severe COVID-19, COVID-19 hospitalization, and SARS-CoV-2 infection with RA, and 19, 24, and 11 shared loci with SLE, respectively. Shared genes were significantly enriched in the spleen, lung, whole blood, and small intestine, and involved in immune function, inflammation and coagulation process. Co-localization analysis identified eight shared loci in TYK2, IKZF3, COL11A2, PSORS1C1, MANEAL and COG6 genes for COVID-19 with RA, and four in CRHR1, FUT2 and NXPE3 genes for COVID-19 with SLE. Bi-directional Mendelian randomization analysis suggested RA is associated with a higher risk of COVID-19 hospitalization, and COVID-19 is not related to RA or SLE. Our novel findings improved the understanding of the common genetic aetiology shared by COVID-19, RA and SLE, and suggested an increased risk of COVID-19 hospitalization in people with higher genetic liability to RA.

An attenuated vaccinia vaccine encoding the SARS-CoV-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human ACE2 transgenic mice from SARS-CoV-2 and its variants (preprint)

As long as the coronavirus disease 2019 (COVID-19) pandemic continues, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with altered antigenicity will emerge. The development of vaccines that elicit robust, broad, and durable protection against SARS-CoV-2 variants is urgently needed. We have developed a vaccine (rDIs-S) consisting of the attenuated vaccinia virus DIs strain platform carrying the SARS-CoV-2 S gene. rDIs-S induced neutralizing antibody and T-lymphocyte responses in cynomolgus macaques and human angiotensin converting enzyme 2 (hACE2) transgenic mice, and showed broad protection against SARS-CoV-2 isolates ranging from the early-pandemic strain (WK-521) to the recent Omicron BA.1 variant (TY38-839). Using a tandem mass tag (TMT) -based quantitative proteomic analysis of lung homogenates from hACE2 transgenic mice, we found that, among mice subjected to challenge infection with WK-521, vaccination with rDIs-S prevented protein expression related to the severe pathogenic effects of SARS-CoV-2 infection (tissue destruction, inflammation, coagulation, fibrosis, and angiogenesis) and restored protein expression related to immune responses (antigen presentation and cellular response to stress). Furthermore, long-term studies in mice showed that rDIs-S maintains S protein-specific antibody titers for at least 6 months after a 1st vaccination. Thus, rDIs-S appears to provide broad and durable protective immunity against SARS-CoV-2, including current and possibly future variants.

Pilot study to evaluate hypercoagulation and inflammation using rotational thromboelastometry and calprotectin in COVID-19 patients. (preprint)

IntroductionAbnormal coagulation and inflammation are hallmarks of SARs-COV-19. Stratifying affected patients on admission to hospital may help identify those who are risk of developing severe disease early on. ROTEM is a point of care test that can be used to measure abnormal coagulation and calprotectin is a measure of inflammation. AimAssess if ROTEM can measure hypercoagulability on admission and identify those who will develop severe disease early on. Assess if calprotectin can measure inflammation and if there is a correlation with ROTEM and calprotectin. MethodsCOVID-19 patients were recruited on admission and ROTEM testing was undertaken daily for a period of 7 days. Additionally inflammatory marker calprotectin was also tested. Results33 patients were recruited to the study out of which 13 were admitted to ITU and 20 were treated on the ward. ROTEM detected a hypercoagulable state on admission but did not stratify between those admitted to a ward or escalated to ITU. Calprotectin levels were raised but there was no statistical difference (p=0.73) between groups. Significant correlations were observed between FIBA5 (p<0.00), FIBCFT (p<0.00), FIBMCF (p<0.00) and INMCF (p<0.00) and calprotectin. ConclusionCOVID19 patients were hypercoagulable in admission. The correlations between ROTEM and calprotectin underline the interactions between inflammation and coagulation.

Shared genetic etiology and causality between COVID-19 and venous thromboembolism: evidence from genome-wide cross trait analysis and bi-directional Mendelian randomization study (preprint)

Venous thromboembolism (VTE) occurs in up to one third patients with COVID-19. VTE and COVID-19 may share a common genetic architecture in etiology, which has not been comprehensively investigated. In this study, we leveraged summary-level data from the latest COVID-19 host genetics consortium and UK Biobank to study the genetic commonality between COVID-19 traits and VTE. We found a positive genetic correlation between COVID-19 hospitalization and VTE (rg = 0.2320, P-value= 0.0092). The cross-trait analysis identified shared genetic loci between VTE and COVID-19 traits, including 8 for severe COVID-19, 11 for COVID-19 hospitalization, and 7 for SARS-CoV-2 infection. We identified seven novel mapped genes (LINC00970, TSPAN15, ADAMTS13, F5, DNAJB4, SLC39A8 and OBSCN) that were enriched for expression in the lung tissue, and in coagulation and immune related pathways. Eight genetic loci were found to share causal variants between COVID-19 and VTE, which are localized in the ABO, ADAMTS13 and FUT2 gene regions. Bi-directional Mendelian randomization analysis did not suggest a causal relationship between VTE and COVID-19 traits. Our study advances the understanding of shared genetic etiology of COVID-19 and VTE at the molecular and functional levels.

Anti-inflammatory therapy with nebulised dornase alfa in patients with severe COVID-19 pneumonia (preprint)

Background: SARS-CoV2 infection causes severe, life-threatening pneumonia. Hyper-inflammation, coagulopathy and lymphopenia are associated with pathology and poor outcomes in these patients. Cell-free (cf) chromatin is prominent in COVID-19 patients, amplifies inflammation and promotes coagulopathy and immune dysfunction. We hypothesized that cf-chromatin clearance by nebulised dornase alfa may reduce inflammation and improve disease outcomes. Here, we evaluated the efficacy of nebulized dornase alfa in patients hospitalised with severe COVID-19 pneumonia. Methods: In this randomised controlled single-centre phase 2 proof-of-concept trial, we recruited adult patients admitted to hospital that exhibited stable oxygen saturation ([≥]94%) on supplementary oxygen and a C-reactive protein (CRP) level [≥]30mg/L post dexamethasone treatment. Participants were randomized at a 3:1 ratio to receive twice-daily nebulised dornase alfa in addition to best available care (BAC) or BAC alone for seven days or until hospital discharge. A 2:1 ratio of historical controls to treated individuals (HC, 2:1) were included as the primary endpoint comparators. The primary outcome was a reduction in systemic inflammation measured by blood CRP levels over 7 days post-randomisation, or to discharge if sooner. Secondary and exploratory outcomes included time to discharge, time on oxygen, D-dimer levels, lymphocyte counts and levels of circulating cf-DNA. Results: We screened 75 patients and enrolled 39 participants out of which 30 in dornase alfa arm, and 9 in BAC group. We also matched the recruited patients in the treated group (N=30) to historical controls in the BAC group (N=60). For the the primary outcome, 30 patients in the dornase alfa were compared to 69 patients in the BAC group. Dornase alfa treatment reduced CRP by 33% compared to the BAC group at 7-days (P=0.01). The dornase alfa group least squares mean CRP was 23.23 mg/L (95% CI 17.71 to 30.46) and the BAC group 34.82 mg/L (95% CI 28.55 to 42.47). A significant difference was also observed when only randomised participants were compared. Furthermore, compared to the BAC group, the chance of live discharge was increased by 63% in the dornase alfa group (HR 1.63, 95% CI 1.01 to 2.61, P=0.03), lymphocyte counts were improved (least-square mean: 1.08 vs 0.87, P=0.02) and markers of coagulopathy such as D-dimer were diminished (least-square mean: 570.78 vs 1656.96g/mL, P=0.004). Moreover, the dornase alfa group exhibited lower circulating cf-DNA levels that correlated with CRP changes over the course of treatment. No differences were recorded in the rates and length of stay in the ICU or the time on oxygen between the groups. Dornase alfa was well-tolerated with no serious adverse events reported. Conclusions: In this proof-of-concept study in patients with severe COVID-19 pneumonia, treatment with nebulised dornase alfa resulted in a significant reduction in inflammation, markers of immune pathology and time to discharge. The effectiveness of dornase alfa in patients with acute respiratory infection and inflammation should be investigated further in larger trials. Trial registration number: NCT04359654

FDP Fluctuation in Severe COVID-19 is Associated With the Development of Thrombotic or Bleeding Complications and Systemic Coagulopathy (preprint)

Purpose: COVID-19 is sometimes associated with coagulation disorders. In such cases, patients developed elevated D‐dimer and fibrin degradation products (FDP) levels, both of which are associated with high risks of thromboembolic complications and poor prognosis. To date, time course changes of FDP values in COVID-19 patients has not been well evaluated. The aim of this study is to evaluate whether FDP fluctuation in COVID-19 patients are associated with systemic coagulopathy. Methods: We retrospectively analyzed the changes in coagulofibrinolytic markers including FDP in 42 COVID-19-ARDS patients. FDP elevation as the fluctuation was defined as follows: 1) FDP>10μg/mL for the first time after admission and 2) 10μg/mL or more elevation after the improvement of the first or subsequent FDP elevations. Results: FDP elevation was observed a total of 30 times in 21 patients (50%). Marked intravascular coagulofibrinolytic activation occurred at the same time as the FDP elevation (soluble fibrin: SF, 27.0 [14.9–80.0] μg/mL; thrombin-antithrombin complex: TAT, 7.5 [2.9–17.8] μg/L; plasmin-α 2 -plasmin inhibitor complex: PIC, 2.4 [1.4–4.2] μg/mL). FDP was elevated in all patients who met sepsis-induced coagulopathy (SIC) or disseminated intravascular coagulation (DIC) diagnosis criteria. Thrombotic or bleeding complications developed in 12 patients (28.6%) and were significantly correlated with FDP elevation ( OR [odds ratio] 4.50, 95% CI [confidence interval] 1.01–20.11, p = 0.049). However, there were no significant differences in coagulofibrinolytic activities between the patients with and without SIC or DIC. Conclusions: Coagulation activation which can lead to the development of systemic coagulopathy such as DIC occurred with FDP fluctuation in severe COVID-19 patients. However, there is a limit of the application of existing DIC and SIC diagnosis criteria to COVID-19.

Functional proteomic profiling links deficient DNA clearance to mortality in patients with severe COVID-19 pneumonia (preprint)

Hyperinflammation, coagulopathy and immune dysfunction are prominent in patients with severe infections. Extracellular chromatin clearance by plasma DNases suppresses such pathologies in mice but whether severe infection interferes with these pathways is unclear. Here, we show that patients with severe SARS-CoV-2 infection or microbial sepsis exhibit low extracellular DNA clearance capacity associated with the release of the DNase inhibitor actin. Unlike naked DNA degradation (DNase), neutrophil extracellular trap degradation (NETase) was insensitive to G-actin, indicating distinct underlying mechanisms. Functional proteomic profiling of severely ill SARS-CoV-2 patient plasma revealed that patients with high NETase and DNase activities exhibited 18-fold higher survival compared to patients with low activity proteomic profiles. Remarkably, low DNA clearance capacity was also prominent in healthy individuals with chronic inflammation, suggesting that pre-existing inflammatory conditions may increase the risk for mortality upon infection. Hence, functional proteomic profiling illustrates that non-redundant DNA clearance activities protect critically ill patients from mortality, uncovering protein combinations that can accurately predict mortality in critically ill patients.

Crosstalk among strenuous exercise, IL-6 and S-Protein Based Vaccines for COVID-19 may explain the rare adverse effects of myocarditis and thrombosis in recently vaccinated young people. A prospective observational study. (preprint)

Interleukin 6 (IL-6) is a type of interleukin that functions as both a pro-inflammatory and anti-inflammatory cytokine. It is encoded by the IL6 gene in humans. Both COVID-19 infection and S-Protein Based Vaccines for COVID-19 were found to induce the production of proinflammatory IL-6, and also, strenuous exercise was found to induce IL-6 secretion by the skeletal muscles via lactate. Exercise causes skeletal muscle cells to release IL-6, and it raises the plasma concentration of IL-6 100 times higher than at rest . Exercise-induced IL-6 release is highly correlated with exercise intensity and duration; thus, IL-6 is regarded as an energy sensor released by contracting muscles. but It was found that rare adverse effects such as myocardial infarction and coagulation abnormalities , are rarely associated with S-Protein Based Vaccines for COVID-19. Recently, it was showed in a study that most patients who experienced myocarditis after COVID-19 vaccine were young male youth aged 16 to 29 years had the highest incidence of myocarditis. Interestingly, It was observed that IL-6 was linked to adverse effects such as thrombosis and myocarditis, both of which are similar to that was caused by COVID-19 infection, and that S-Protein Based Vaccines for COVID-19. Here, we propose a testable hypothesis that strenuous exercise could be a risk and cofactor helping in the existing of these adverse effects in young people such as myocarditis and thrombosis via induction the secretion of proinflammatory IL-6. In our prospective observational study we will assess the possible correlation among the strenuous exercise , IL-6, myocarditis and thrombosis. The study will be multi-center and will involve young patient who will be vaccinated with first ,second and third dose of S-Protein Based Vaccines for COVID-19 (Moderna and Pfizer-BioNTech vaccine). Pfizer and BioNTech have successfully developed the BNT162b2 mRNA vaccine, which consists of the full-length S glycoprotein with the K986P and V987P mutation sites. Also, Moderna's mRNA-1273 vaccine contains the coding sequence for a S glycoprotein stabilized by a pair of proline substitutions (K986P/V987P), a transmembrane anchor, and an intact S1-S2 cleavage site

GB0139, an inhaled small molecule inhibitor of galectin-3, in COVID-19 pneumonitis: a randomised, controlled, open-label, phase 2a experimental medicine trial of the safety, pharmacokinetics, and potential therapeutic value (preprint)

Rationale: High galectin-3 levels predict poor outcomes in patients with COVID-19. Galectin-3 activates monocytes and macrophages which are directly implicated in COVID-19 immunopathology and the cytokine storm. GB0139 is a potent thiodigalactoside galectin-3 inhibitor and may reduce the severe effects of the disease. We report safety and pharmacokinetics and pharmacodynamics of the inhaled galectin-3 inhibitor, GB0139, and assess clinical outcomes and key systemic inflammatory biomarkers in hospitalised patients with COVID-19 (ClinicalTrials.gov/EudraCT identifier: NCT04473053/2020-002230-32). Methods: Adults with COVID-19 requiring oxygen, and with pneumonitis on x-ray, were randomised to receive standard of care (SOC; including dexamethasone; n=21) or SOC plus 10 mg GB0139 twice daily for 48 hours, then once daily for [≤]14 days (n=20). Results: Patients aged 27-87 years were enrolled from July 2020; the final patient completed the 90-day follow-up in April 2021. GB0139+SOC was well tolerated with no treatment-related serious adverse events reported. Incidences of adverse events were similar between treatment arms (40 with GB0139+SOC vs 35 with SOC). Plasma GB0139 was measurable in all patients after inhaled exposure, with moderate interpatient variability, and demonstrated target engagement with decreased circulating galectin (overall treatment effect post-hoc over days 2-7: p=0{middle dot}0099 vs SOC). Rate of decline in fraction of inspired oxygen (%) requirement was significantly greater in the GB0139+SOC arm with a posterior mean difference of -1 {middle dot}51 (95% highest posterior density: -2{middle dot}90, -0{middle dot}189) versus SOC. Plasma levels of biomarkers associated with inflammation, coagulopathy, major organ function and fibrosis showed a downward trend versus SOC. Conclusions: GB0139+SOC was well tolerated and achieved clinically relevant plasma concentrations and target engagement. This, and the reduction in markers associated with inflammatory, coagulation, fibrosis, and reduction in inspired oxygen (%) over SOC alone, indicates the therapeutic potential for inhaled GB0139 in hospitalised patients with COVID-19.

Long Covid after Breakthrough COVID-19: the post-acute sequelae of breakthrough COVID-19 (preprint)

The post-acute sequelae of COVID-19 have been described 1 , but whether breakthrough COVID-19 (that is the disease that ensues following vaccine breakthrough SARS-CoV-2 infection) results in post-acute sequelae is not yet clear. Here we use the national healthcare databases of the US Department of Veterans Affairs to characterize 6-month risks of incident post-acute sequelae in people with breakthrough COVID-19 who survived for at least 30 days after diagnosis. We show that compared to people with no evidence of COVID-19, beyond the first 30 days of illness, people with breakthrough COVID-19 exhibit a higher risk of death and broad array of incident post-acute sequelae in the pulmonary system, as well as extrapulmonary sequelae that include cardiovascular disorders, coagulation disorders, gastrointestinal disorders, general disorders (e.g., fatigue), kidney disorders, mental health disorders, metabolic disorders, musculoskeletal disorders, and neurologic disorders. Our analyses by care setting of the acute phase of the disease show that people who were not hospitalized during the first 30 days after diagnosis with breakthrough COVID-19 exhibit a small but not insignificant increase in risk of death and post-acute sequelae; the risks are further increased in people who were hospitalized during the acute phase of the disease. Our comparative approach shows that people with breakthrough COVID-19 exhibit lower risks of death and post-acute sequelae than people with COVID-19 who were not previously vaccinated for it; and in analyses among individuals who were hospitalized during the acute phase of the disease, people with breakthrough COVID-19 exhibit higher risks of death and post-acute sequelae than people with seasonal influenza. Altogether, our findings show increased risks of death and post-acute sequalae in people with breakthrough COVID-19; the risks are evident among those who were not hospitalized during the acute phase of the disease. Our comparative approach provides context for understanding the risks in relation to COVID-19 without prior vaccination and seasonal influenza. The findings will inform the ongoing effort to optimize strategies for prevention of breakthrough SARS-CoV-2 infections and will guide development and optimization of post-acute care pathways for people with breakthrough COVID-19.

COVID-19 is associated with higher risk of venous thrombosis, but not arterial thrombosis, compared with influenza: Insights from a large US cohort (preprint)

Introduction Infection with SARS-CoV-2 is typically compared with influenza to contextualize its health risks. SARS-CoV-2 has been linked with coagulation disturbances including arterial thrombosis, leading to considerable interest in antithrombotic therapy for Coronavirus Disease 2019 (COVID-19). However, the independent thromboembolic risk of SARS-CoV-2 infection compared with influenza remains incompletely understood. We evaluated the adjusted risks of thromboembolic events after a diagnosis of COVID-19 compared with influenza in a large retrospective cohort. Methods We used a US-based electronic health record (EHR) dataset linked with insurance claims to identify adults diagnosed with COVID-19 between April 1, 2020 and October 31, 2020. We identified influenza patients diagnosed between October 1, 2018 and April 31, 2019. Primary outcomes [venous composite of pulmonary embolism (PE) and acute deep vein thrombosis (DVT); arterial composite of ischemic stroke and myocardial infarction (MI)] and secondary outcomes were assessed 90 days post-diagnosis. Propensity scores (PS) were calculated using demographic, clinical, and medication variables. PS-adjusted hazard ratios (HRs) were calculated using Cox proportional hazards regression. Results There were 417,975 COVID-19 patients (median age 57y, 61% women), and 345,934 influenza patients (median age 47y, 66% women). Compared with influenza, patients with COVID-19 had higher venous thromboembolic risk (HR 1.53, 95% CI 1.38-1.70), but not arterial thromboembolic risk (HR 1.02, 95% CI 0.95-1.10). Secondary analyses demonstrated similar risk for ischemic stroke (HR 1.11, 95% CI 0.98-1.25) and MI (HR 0.93, 95% CI 0.85-1.03) and higher risk for DVT (HR 1.36, 95% CI 1.19-1.56) and PE (HR 1.82, 95% CI 1.57-2.10) in patients with COVID-19. Conclusion In a large retrospective US cohort, COVID-19 was independently associated with higher 90-day risk for venous thrombosis, but not arterial thrombosis, as compared with influenza. These findings may inform crucial knowledge gaps regarding the specific thromboembolic risks of COVID-19.

Gene Expression Risk Scores for COVID-19 Illness Severity (preprint)

BackgroundThe correlates of COVID-19 illness severity following infection with SARS-Coronavirus 2 (SARS-CoV-2) are incompletely understood. MethodsWe assessed peripheral blood gene expression in 53 adults with confirmed SARS-CoV-2-infection clinically adjudicated as having mild, moderate or severe disease. Supervised principal components analysis was used to build a weighted gene expression risk score (WGERS) to discriminate between severe and non-severe COVID. ResultsGene expression patterns in participants with mild and moderate illness were similar, but significantly different from severe illness. When comparing severe versus non-severe illness, we identified >4000 genes differentially expressed (FDR<0.05). Biological pathways increased in severe COVID-19 were associated with platelet activation and coagulation, and those significantly decreased with T cell signaling and differentiation. A WGERS based on 18 genes distinguished severe illness in our training cohort (cross-validated ROC-AUC=0.98), and need for intensive care in an independent cohort (ROC-AUC=0.85). Dichotomizing the WGERS yielded 100% sensitivity and 85% specificity for classifying severe illness in our training cohort, and 84% sensitivity and 74% specificity for defining the need for intensive care in the validation cohort. ConclusionThese data suggest that gene expression classifiers may provide clinical utility as predictors of COVID-19 illness severity.