Fluoxetine and Sertraline Potently Neutralize the Replication of Distinct SARS-CoV-2 Variants (preprint)

The pandemic caused by SARS-CoV-2 is still a major health problem. Newly emerging variants and long-COVID-19 represent a challenge for the global health system. In particular, individuals in developing countries with insufficient health care need easily accessible, affordable and effective treatments of COVID-19. Previous studies have demonstrated the efficacy of functional inhibitors of acid sphingomyelinase (FIASMA) against infections with various viruses, including early variants of SARS-CoV-2. This work investigated whether the acid sphingomyelinase inhibitors fluoxetine and sertraline, usually used as antidepressant molecules in clinical practice, can inhibit the replication of the former and recently emerged SARS-CoV-2 variants in vitro. Fluoxetine and sertraline potently inhibited the infection with pseudotyped virus like particles and SARS-CoV-2 variants D614G, alpha, delta, omicron BA.1 and omicron BA.5. These results highlight fluoxetine and sertraline as priority candidates for large-scale phase 3 clinical trials at different stages of SARS-CoV-2 infections, either alone or in combination with other medications.

Fading SARS-CoV-2 humoral VOC cross-reactivity versus sustained cellular immunity in convalescent children and adolescents (preprint)

Cross-reactive cellular and humoral immunity can substantially contribute to antiviral defense against SARS-CoV-2 variants of concern (VOC). While the adult SARS-CoV-2 cellular and humoral immunity and its cross-recognition potential against VOC is broadly analyzed, similar data regarding the pediatric population are missing. In this study, we perform an analysis of the humoral and cellular SARS-CoV-2 response immune of 32 convalescent COVID-19 children (children), 27 convalescent vaccinated adults(C+V+) and 7 unvaccinated convalescent adults (C+V-). SARS-CoV-2 reactive T cell response is analyzed via multiparametric flowcytometry and humoral immunity is addressed via pseudovirus neutralization assay. Similarly to adults, a significant reduction of cross-reactive neutralizing capacity against delta and omicron VOC was observed 6 months after SARS-CoV-2 infection. While SAR-CoV-2 neutralizing capacity was comparable among children and C+V- against all VOC, children demonstrated as expected an inferior humoral response when compared to C+V+. Nevertheless, children generated SARS-CoV-2 reactive T cells with broad cross-recognition potential. When compared to V+C+, children presented even comparable frequencies of WT-reactive CD4+ and CD8+ T cells with high avidity. Our results suggest that following SARS-CoV-2 infection children generate a humoral SARS-CoV-2 response with neutralizing potential comparable to unvaccinated COVID-19 convalescent adults as well a sustained SARS-CoV-2 cellular response cross-reactive to VOC. Key statement Following SARS-CoV-2 infection children generate a humoral SARS-CoV-2 response with neutralizing potential comparable to unvaccinated COVID-19 convalescent adults as well a sustained robust SARS-CoV-2 cellular response cross-reactive to VOC, comparable to vaccinated convalescent adults.

Increased migratory/activated CD8+ T cell and low avidity SARS-CoV-2 reactive cellular response in post-acute COVID-19 syndrome (preprint)

The role of autoimmunity in post-acute sequelae of COVID-19 (PASC) is not well explored, although clinicians observe a growing population of convalescent COVID-19 patients with manifestation of post-acute sequelae of COVID-19. We analyzed the immune response in 40 post-acute sequelae of COVID-19 patients with non-specific PASC manifestation and 15 COVID-19 convalescent healthy donors. The phenotyping of lymphocytes showed a significantly higher number of CD8+ T cells expressing the Epstein-Barr virus induced G protein coupled receptor 2, chemokine receptor CXCR3 and C-C chemokine receptor type 5 playing an important role in inflammation and migration in PASC patients compared to controls. Additionally, a stronger, SARS-CoV-2 reactive CD8+ T cell response, characterized by IFN{gamma} production and predominant TEMRA phenotype but low SARS-CoV-2 avidity was detected in PASC patients compared to controls. Furthermore, higher titers of several autoantibodies were detected among PASC patients. Our data suggest that a persistent inflammatory response triggered by SARS-CoV-2 might be responsible for the observed sequelae in PASC patients. These results may have implications on future therapeutic strategies.

Anti-SARS-CoV-2 antibody containing plasma improves outcome in patients with hematologic or solid cancer and severe COVID-19 via increased neutralizing antibody activity. A randomized clinical trial (preprint)

Cancer patients are at high risk of severe COVID-19 with high morbidity and mortality. Further, impaired humoral response renders SARS-CoV-2 vaccines less effective and treatment options are scarce. Randomized trials using convalescent plasma are missing for high-risk patients. Here, we performed a multicenter trial (https://www.clinicaltrialsregister.eu/ctr-search/trial/2020-001632-10/DE) in hospitalized patients with severe COVID-19 within four risk groups (1, cancer; 2, immunosuppression; 3, lab-based risk factors; 4, advanced age) randomized to standard of care (CONTROL) or standard of care plus convalescent/vaccinated anti-SARS-CoV-2 plasma (PLASMA). For the four groups combined, PLASMA did not improve clinically compared to CONTROL (HR 1.29; p=0.205). However, cancer patients experienced shortened median time to improvement (HR 2.50, p=0.003) and superior survival in PLASMA vs. CONTROL (HR 0.28; p=0.042). Neutralizing antibody activity increased in PLASMA but not in CONTROL cancer patients (p=0.001). Taken together, convalescent/vaccinated plasma may improve COVID-19 outcome in cancer patients unable to intrinsically generate an adequate immune response.

New susceptibility loci for severe COVID-19 by detailed GWAS analysis in European populations (preprint)

Due to the highly variable clinical phenotype of Coronavirus disease 2019 (COVID-19), deepening the host genetic contribution to severe COVID-19 may further improve our understanding about underlying disease mechanisms. Here, we describe an extended GWAS meta-analysis of 3,260 COVID-19 patients with respiratory failure and 12,483 population controls from Italy, Spain, Norway and Germany, as well as hypothesis-driven targeted analysis of the human leukocyte antigen (HLA) region and chromosome Y haplotypes. We include detailed stratified analyses based on age, sex and disease severity. In addition to already established risk loci, our data identify and replicate two genome-wide significant loci at 17q21.31 and 19q13.33 associated with severe COVID-19 with respiratory failure. These associations implicate a highly pleiotropic ~0.9-Mb 17q21.31 inversion polymorphism, which affects lung function and immune and blood cell counts, and the NAPSA gene, involved in lung surfactant protein production, in COVID-19 pathogenesis.

Generation of Inhibitory Autoantibodies to ADAMTS13 in Coronavirus Disease 2019 (preprint)

Objectives: It has recently been shown that von Willebrand factor (vWf) multimers may be a key driver of immunothrombosis in Coronavirus disease 2019 (COVID-19). Since COVID-19 is associated with an increased risk of autoreactivity, the present study investigates, whether the generation of autoantibodies to ADAMTS13 contributes to this finding. Design: Observational prospective controlled multicenter study. Setting: Blood samples and clinical data of patients with COVID-19 were collected regularly during hospitalization in the period from April to November 2020. Patients: 90 patients with confirmed COVID-19 of mild to critical severity and 30 healthy controls participated in this study. Measuerements and Main Results: Antibodies to ADAMTS13 occurred in 31 (34.4%) patients with COVID-19. Generation of ADAMTS13 antibodies was associated with a significantly lower ADAMTS13 activity (56.5%, interquartile range (IQR) 21.25 vs. 71.5%, IQR 24.25, p=0.0041), increased disease severity (severe or critical disease in 90% vs. 62.3%, p=0.0189), and a trend to a higher mortality (35.5% vs. 18.6%, p=0.0773). Median time to antibody development was 11 days after first positive SARS-CoV-2-PCR specimen. Conclusion: The present study demonstrates for the first time, that generation of antibodies to ADAMTS13 is a frequent finding in COVID-19. Generation of these antibodies is associated with a lower ADAMTS13 activity and an increased risk of an adverse course of the disease suggesting an inhibitory effect on the protease. These findings provide a rationale to include ADAMTS13 antibodies in the diagnostic workup of SARS-CoV-2 infections.

Disinfection of SARS-CoV-2 contaminated surfaces of personal items with UVC-LED disinfection boxes (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is transmitted from person-to-person by close contact, small aerosol respiratory droplets and potentially via contact with contaminated surfaces. Here, we investigated the effectiveness of commercial UVC-LED disinfection boxes in inactivating SARS-CoV-2 contaminated surfaces of personal items. We contaminated glass, metal and plastic samples representing the surfaces of personal items such as smartphones, coins or credit cards with SARS-CoV-2 formulated in an organic matrix mimicking human respiratory secretions. For disinfection, the samples were placed at different distances from UVC emitting LEDs inside commercial UVC-LED disinfection boxes and irradiated for different time periods (up to 10 minutes). High viral loads of SARS-CoV-2 were effectively inactivated on all surfaces after 3 minutes of irradiation. Even 10 seconds of UVC-exposure strongly reduced viral loads. Thus, UVC-LED boxes proved to be an effective method for disinfecting SARS-CoV-2 contaminated surfaces that are typically found on personal items.

Detection of SARS-CoV-2 Specific Memory B cells to Delineate Long-Term COVID-19 Immunity (preprint)

Background: The ongoing COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, represents a serious worldwide health concern. A deeper understanding of the immune response to SARS-CoV-2 will be required to refine vaccine development and efficacy as well as to evaluate long-term immunity in convalescent patients. With this in mind, we investigated the formation of SARS-CoV-2 specific BMEMORY cells from patient blood samples. Methods: A standard flow cytometry-based protocol for the detection of SARS-CoV-2 specific B cells was applied using fluorochrome-coupled SARS-CoV-2 spike (S) full-length protein. Cohorts of 26 central European convalescent mild/moderate COVID-19 patients and 14 healthy donors were assessed for the levels of SARS-CoV-2 S- specific BMEMORY cells. Results: Overall B cell composition was not affected by SARS-CoV-2 infection in convalescent patients. Our analysis of SARS-CoV-2 specific BMEMORY cells in samples collected at different time points revealed that S-protein specific B cells remain in peripheral blood at least up to 6 months after COVID-19 diagnosis. Conclusions: Detection of SARS-CoV-2 specific BMEMORY cells may improve our understanding of the long-term adaptive immunity in response to SARS-CoV-2, allowing for an improved public health response and vaccine development during the COVID-19 pandemic. Further validation of the study in larger and more diverse populations and a more extended observation period will be required.

Glycyrrhizin effectively neutralizes SARS-CoV-2 in vitro by inhibiting the viral main protease (preprint)

The newly emerged coronavirus, which was designated as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 disease. High effective and well-tolerated medication for hospitalized and non-hospitalized patients is urgently needed. Traditional herbal medicine substances were discussed as promising candidates for the complementary treatment of viral diseases and recently suggested for the treatment of COVID-19. In the present study, we investigated aqueous licorice root extract for its neutralizing activity against SARS-CoV-2 in vitro, identified the active compound glycyrrhizin and uncovered the respective mechanism of viral neutralization. We demonstrated that glycyrrhizin, the primary active ingredient of the licorice root, potently neutralizes SARS-CoV-2 by inhibiting the viral main protease. Our experiments highlight glycyrrhizin as a potential antiviral compound that should be further investigated for the treatment of COVID-19.

SARS-CoV-2-specific humoral and cellular immunity in renal transplant and haemodialysis patients treated with convalescent plasma (preprint)

BackgroundWhen patients with chronic kidney disease are infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) they can face two specific problems: Virus-specific immune responses may be impaired and remdesivir, an antiviral drug described to shorten the time to recovery, is contraindicated. Antiviral treatment with convalescent plasma could be an alternative treatment option. MethodsIn this case series we present two kidney transplant recipients and two patients dependent on haemodialysis who were infected with SARS-CoV-2 and received convalescent plasma. Antibodies against the spike 1 protein of SARS-CoV-2 were determined sequentially by IgG ELISA and neutralization assay and specific T cell responses by interferon-gamma ELISpot. ResultsPrior to treatment, in three patients antibodies were undetectable by ELISA (ratio < 1.1), corresponding to low neutralizing antibody titers ([≤] 1:40). One patient was also negative to the ELISpot and two showed weak responses. After convalescent plasma treatment we observed an increase of SARS-CoV-2-specific antibodies (IgG ratio and neutralization titer) and of specific T cell responses. After intermittent clinical improvement one kidney transplant recipient again developed typical symptoms at day 12 after treatment and received a second cycle of convalescent plasma treatment. Altogether, three patients clinically improved and could be discharged from hospital. However, one multimorbid female in her early eighties deceased. ConclusionsOur data suggest that the success of convalescent plasma therapy may only be temporary in patients with chronic kidney disease; which requires an adaptation of the treatment regimen. Close monitoring after treatment is needed for this patient group.

COVID-19 is associated with relative ADAMTS13 deficiency and VWF multimer formation resembling TTP (preprint)

Background: Thrombotic microangiopathy (TMA) has been repeatedly described in COVID-19 and may contribute to SARS-CoV-2 associated hypercoagulability. The underlying mechanisms remain elusive. We hypothesized that endothelial damage may lead to substantially increased concentrations of Von Willebrand Factor (VWF) with subsequent relative deficiency of ADAMTS13. Methods: A prospective controlled trial was performed on 75 patients with COVID-19 of mild to critical severity and 10 healthy controls. VWF antigen (VWF:Ag), ADAMTS13 and VWF multimer formation were analyzed in a German hemostaseologic laboratory. Results: VWF:Ag was 4.8 times higher in COVID-19 patients compared to healthy controls (p<0.0001), whereas ADAMTS13 activities were not significantly different (p=0.24). The ADAMTS13/VWF:Ag ratio was significantly lower in COVID-19 than in the control group (24.4{+/-}20.5 vs. 79.7{+/-}33.2, p<0.0001). Fourteen patients (18.7%) undercut a critical ratio of 10 as described in thrombotic thrombocytopenic purpura (TTP). Gel analysis of multimers resembled the TTP constellation with loss of the largest multimers in 75% and a smeary triplet pattern in 39% of the patients. The ADAMTS13/VWF:Ag ratio decreased continuously from mild to critical disease (ANOVA p=0.026). Moreover, it differed significantly between surviving patients and those who died from COVID-19 (p=0.001) yielding an AUC of 0.232 in ROC curve analysis. Conclusion: COVID-19 is associated with a substantial increase in VWF levels, which can exceed the ADAMTS13 processing capacity resulting in the formation of large VWF multimers identical to TTP. The ADAMTS13/VWF:Ag ratio is an independent predictor of severity of disease and mortality. These findings render further support to perform studies on the use of plasma exchange in COVID-19 and to include VWF and ADAMTS13 in the diagnostic workup.

Impaired cytotoxic CD8+ T cell response in elderly COVID-19 patients (preprint)

SARS-CoV-2 infection induces a T cell response that most likely contributes to virus control in COVID-19 patients, but may also induce immunopathology. Until now, the cytotoxic T cell response has not been very well characterized in COVID-19 patients. Here, we analyzed the differentiation and cytotoxic profile of T cells in 30 cases of mild COVID-19 during acute infection. SARS-CoV-2 infection induced a cytotoxic response of CD8+ T cells, but not CD4+ T cells, characterized by the simultaneous production of granzyme A and B, as well as perforin within different effector CD8+ T cell subsets. PD-1 expressing CD8+ T cells also produced cytotoxic molecules during acute infection indicating that they were not functionally exhausted. However, in COVID-19 patients over the age of 80 years the cytotoxic T cell potential was diminished, especially in effector memory and terminally differentiated effector CD8+ cells, showing that elderly patients have impaired cellular immunity against SARS-CoV-2. Our data provides valuable information about T cell responses in COVID-19 patients that may also have important implications for vaccine development. ImportanceCytotoxic T cells are responsible for the elimination of infected cells and are key players for the control of viruses. CD8+ T cells with an effector phenotype express cytotoxic molecules and are able to perform target cell killing. COVID-19 patients with a mild disease course were analyzed for the differentiation status and cytotoxic profile of CD8+ T cells. SARS-CoV-2 infection induced a vigorous cytotoxic CD8+ T cell response. However, this cytotoxic profile of T cells was not detected in COVID-19 patients over the age of 80 years. Thus, the absence of a cytotoxic response in elderly patients might be a possible reason for the more frequent severity of COVID-19 in this age group in comparison to younger patients.

Antiviral activity of lambda-carrageenan against influenza viruses in mice and severe acute respiratory syndrome coronavirus 2 in vitro (preprint)

Influenza virus and coronavirus, belonging to enveloped RNA viruses, are major causes of human respiratory diseases. The aim of this study was to investigate the broad spectrum antiviral activity of a naturally existing sulfated polysaccharide, lambda-carrageenan ({lambda}-CGN), purified from marine red algae. Cell culture-based assays revealed that the macromolecule efficiently inhibited both influenza A and B viruses, as well as currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with EC50 values ranging from 0.3-1.4 g/ml. No toxicity to host cells was observed at concentrations up to 300 g/ml. Plaque titration and western blot analysis verified that {lambda}-CGN reduced expression of viral proteins in cell lysates and suppressed progeny virus production in culture supernatants in a dose-dependent manner. This polyanionic compound exerts antiviral activity by targeting viral attachment to cell surface receptors and preventing entry. Moreover, intranasal administration to mice during influenza A viral challenge not only alleviated infection-mediated reductions in body weight but also protected 60% of mice from virus-induced mortality. Thus, {lambda}-CGN could be a promising antiviral agent for preventing infection by several respiratory viruses.

Assessing SARS-CoV-2 RNA levels and lymphocyte/T cell counts in COVID-19 patients revealed initial immune status as a major determinant of disease severity (preprint)

The magnitude of SARS-CoV-2 infection, the dynamic changes of immune parameters in patients with the novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. The clinical and laboratory results from 154 confirmed COVID-19 patients were collected. The SARS-CoV-2 RNA levels in patients were estimated using the Ct values of specific RT-PCR tests. The lymphocyte subsets and cytokines profiles in the peripheral blood were analyzed by flow cytometry and specific immunoassays. 154 confirmed COVID-19 patients were clinically examined up to 4 weeks after admission. The initial SARS-CoV-2 RNA Ct values at admission varied but were comparable in the patient groups classified according to the age, gender, underlying diseases, and disease severity. Three days after admission significant higher Ct values were found in severe cases. Significantly reduced counts of T cells and T cell subsets were found in patients with old age and underlying diseases at admission and were characteristic for the development of severe COVID-19. Severe COVID-19 developed preferentially in patients with underlying compromised immunity and was not associated with initial virus levels. Higher SARS-CoV-2 RNA levels in severe cases were apparently a result of impaired immune control associated with dysregulation of inflammation.

The SARS-CoV-2 T-cell immunity is directed against the spike, membrane, and nucleocapsid protein and associated with COVID 19 severity (preprint)

Identification of immunogenic targets of SARS-CoV-2 is crucial for monitoring of antiviral immunity and vaccine design. Currently, mainly anti-spike (S)-protein adaptive immunity is investigated. However, also the nucleocapsid (N)- and membrane (M)-proteins should be considered as diagnostic and prophylactic targets. The aim of our study was to explore and compare the immunogenicity of SARS-CoV-2 S-, M- and N-proteins in context of different COVID-19 manifestations. Analyzing a cohort of COVID-19 patients with moderate, severe, and critical disease severity, we show that overlapping peptide pools (OPP) of all three proteins can activate SARS-CoV-2-reactive T-cells with a stronger response of CD4+ compared to CD8+ T-cells. Although interindividual variations for the three proteins were observed, M-protein induced the highest frequencies of CD4+ T-cells, suggesting its relevance as diagnostic and vaccination target. Importantly, patients with critical COVID-19 demonstrated the strongest T-cell response, including the highest frequencies of cytokine-producing bi- and trifunctional T-cells, for all three proteins. Although the higher magnitude and superior functionality of SARS-CoV-2-reactive T-cells in critical patients can also be a result of a stronger immunogenicity provided by severe infection, it disproves the hypothesis of insufficient SARS-CoV-2-reactive immunity in critical COVID-19. To this end, activation of effector T-cells with differentiated memory phenotype found in our study could cause hyper-reactive response in critical cases leading to immunopathogenesis. Conclusively, since the S-, M-, and N-proteins induce T-cell responses with individual differences, all three proteins should be evaluated for diagnostics and therapeutic strategies to avoid underestimation of cellular immunity and to deepen our understanding of COVID-19 immunity.

A possible role of immunopathogenesis in COVID-19 progression (preprint)

Background: The efficacy of the humoral and cellular immunity determines the outcome of viral infections. An appropriate immune response mediates protection, whereas an overwhelming immune response has been associated with immune-mediated pathogenesis in viral infections. The current study explored the general and SARS-CoV-2 specific cellular and humoral immune status in patients with different COVID-19 severities. Methods: In this prospective study, we included 53 patients with moderate, severe, and critical COVID-19 manifestations comparing their quantitative, phenotypic, and functional characteristics of circulating immune cells, SARS-CoV-2 antigen specific T-cells, and humoral immunity. Results: Significantly diminished frequencies of CD8+T-cells, CD4+ and CD8+T-cell subsets with activated differentiated memory/effector phenotype and migratory capacity were found in circulation in patients with severe and/or critical COVID-19 as compared to patients with moderate disease. Importantly, the improvement of the clinical courses from severe to moderate was accompanied by an improvement in the T-cell subset alterations. Furthermore, we surprisingly observed a detectable SARS-CoV-2-reactive T-cell response in all three groups after stimulation with SARS-CoV-2 S-protein overlapping peptide pool already at the first visit. Of note, patients with a critical COVID-19 demonstrated a stronger response of SARS-CoV-2-reactive T-cells producing Th1 associated inflammatory cytokines. Furthermore, clear correlation between antibody titers and SARS-CoV-2-reactive CD4+ frequencies underscore the role of specific immunity in disease progression. Conclusion: Our data demonstrate that depletion of activated memory phenotype circulating T-cells and a strong SARS-CoV-2-specific cellular and humoral immunity are associated with COVID-19 disease severity. This counter-intuitive finding may have important implications for diagnostic, therapeutic and prophylactic COVID-19 management.