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.

Impaired humoral immunity to BQ.1.1 in convalescent and vaccinated patients (preprint)

Determining SARS-CoV-2 immunity is critical to assess COVID-19 risk and the need for prevention and mitigation strategies. We measured SARS-CoV-2 Spike/Nucleocapsid seroprevalence and serum neutralizing activity against Wu01, BA.4/5 and BQ.1.1 in 1,411 individuals who received medical treatment in five emergency departments in North Rhine-Westphalia, Germany. We detected Spike-IgG in 95.6%, Nucleocapsid-IgG in 24.0% and neutralization against Wu01, BA.4/5 and BQ.1.1 in 94.4%, 85.0%, and 73.8% of participants, respectively. Neutralization against BA.4/5 and BQ.1.1 was reduced 5.6- and 23.4-fold compared to Wu01. Accuracy of S-IgG detection for determination of neutralizing activity against BQ.1.1 was reduced substantially. Furthermore, we explored previous vaccinations and infections as most important correlates of improved BQ.1.1 neutralization using multivariable and Bayesian network analyses. Given an adherence to COVID-19 vaccination recommendations of only 67.7% of all participants, we highlight the need for improvement of vaccine-uptake to reduce the COVID-19 risk in upcoming infection-waves with immune evasive variants.

Differential interferon-α subtype immune signatures suppress SARS-CoV-2 infection (preprint)

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies and have been successfully employed for the treatment of viral diseases. Humans express twelve IFN-alpha () subtypes, which activate downstream signalling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in type I IFN immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19, therefore, early administration of type I IFNs may be protective against life-threatening disease. Here we comprehensively analysed the antiviral activity of all IFN subtypes against SARS-CoV-2 to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFN subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate and low antiviral IFNs. In particular IFN5 showed superior antiviral activity against SARS-CoV-2 infection. Dose-dependency studies further displayed additive effects upon co-administered with the broad antiviral drug remdesivir in cell culture. Transcriptomics of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting and prototypical genes of individual IFN subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in type I IFN signalling pathways, negative regulation of viral processes and immune effector processes for the potent antiviral IFN5. Taken together, our data provide a systemic, multi-modular definition of antiviral host responses mediated by defined type I IFNs. This knowledge shall support the development of novel therapeutic approaches against SARS-CoV-2.

Comparable environmental stability and disinfection profiles of the currently circulating SARS-CoV-2 variants of concern B.1.1.7 and B.1.351 (preprint)

The emergence of novel SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern with increased transmission dynamics has raised questions regarding stability and disinfection of these viruses. In this study, we analyzed surface stability and disinfection of the currently circulating SARS-CoV-2 variants B.1.1.7 and B.1.351 compared to the wildtype. Treatment with heat, soap and ethanol revealed similar inactivation profiles indicative of a comparable susceptibility towards disinfection. Furthermore, we observed comparable surface stability on steel, silver, copper and face masks. Overall, our data support the application of currently recommended hygiene concepts to minimize the risk of B.1.1.7 and B.1.351 transmission.

Occurrence of COVID-19 symptoms during SARS-CoV-2 infection defines waning of humoral immunity (preprint)

Approximately half of the SARS-CoV-2 infections occur without apparent symptoms, raising questions regarding long-term humoral immunity in asymptomatic individuals. Plasma levels of immunoglobulin G (IgG) and M (IgM) against the viral spike or nucleoprotein were determined for 25,091 individuals enrolled in a surveillance program in Wuhan, China. We compared 405 asymptomatic individuals with 459 symptomatic COVID-19 patients. The well-defined duration of the SARS-CoV-2 endemic in Wuhan allowed a side-by-side comparison of antibody responses following symptomatic and asymptomatic infections without subsequent antigen re-exposure. IgM responses rapidly declined in both groups. However, both the prevalence and durability of IgG responses and neutralizing capacities correlated positively with symptoms. Regardless of sex, age, and body weight, asymptomatic individuals lost their SARS-CoV-2-specific IgG antibodies more often and rapidly than symptomatic patients. These findings have important implications for immunity and favour immunization programs including individuals after asymptomatic infections. One-Sentence SummaryPrevalence and durability of SARS-CoV-2-specific IgG responses and neutralizing capacities correlate with COVID-19 symptoms.

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.

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.

Universally available herbal teas based on sage and perilla elicit potent antiviral activity against SARS-CoV-2 in vitro (preprint)

The current SARS-CoV-2/COVID-19 pandemic represents an unprecedented medical and socioeconomic crisis. Highly efficient treatment options preventing morbidity and mortality are not broadly available and approved drugs are hardly affordable in developing countries. Even after vaccine approvals, it will take several months until the vaccinated and convalescent individuals establish herd immunity. Meanwhile, non-pharmaceutical interventions and antiviral treatments are indispensable to curb the death toll of the pandemic. To identify cost-effective and ubiquitously available options, we tested common herbs consumed worldwide as herbal teas. We found that aqueous infusions prepared by boiling leaves of the Lamiaceae plants perilla and sage elicit potent antiviral activity against SARS-CoV-2 in human cells. Sustained antiviral activity was evident even when cells were treated for only half an hour, and in therapeutic as well as prophylactic regimens. Given the urgency, such inexpensive and broadly available substances might provide help during the pandemic - especially in low-income regions.

The analysis of the long-term impact of SARS-CoV-2 on the cellular immune system in individuals recovering from COVID-19 reveals a profound NKT cell impairment (preprint)

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects millions of people and killed hundred-thousands of individuals. While acute and intermediate interactions between SARS-CoV-2 and the immune system have been studied extensively, long-term impacts on the cellular immune system remained to be analyzed. Here, we comprehensively characterized immunological changes in peripheral blood mononuclear cells in 49 COVID-19 convalescent individuals (CI) in comparison to 27 matched SARS-CoV-2 unexposed individuals (UI). Despite recovery from the disease for more than 2 months, CI showed significant decreases in frequencies of invariant NKT and NKT-like cells compared to UI. Concomitant with the decrease in NKT-like cells, an increase in the percentage of Annexin V and 7-AAD double positive NKT-like cells was detected, suggesting that the reduction in NKT-like cells results from cell death months after recovery. Significant increases in regulatory T cell frequencies, TIM-3 expression on CD4 and CD8 T cells, as well as PD-L1 expression on B cells were also observed in CI, while the cytotoxic potential of T cells and NKT-like cells, defined by GzmB expression, was significantly diminished. However, both CD4 and CD8 T cells of CI showed increased Ki67 expression and were fully capable to proliferate and produce effector cytokines upon TCR stimulation. Collectively, we provide the first comprehensive characterization of immune signatures in patients recovering from SARS-CoV-2 infection, suggesting that the cellular immune system of COVID-19 patients is still under a sustained influence even months after the recovery from disease.

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.

SARS-CoV-2 infection induces sustained humoral immune responses in convalescent patients following symptomatic COVID-19 (preprint)

Long-term antibody responses and neutralizing activities following SARS-CoV-2 infections have not yet been elucidated. We quantified immunoglobulin M (IgM) and G (IgG) antibodies recognizing the SARS-CoV-2 receptor-binding domain (RBD) of the spike (S) or the nucleocapsid (N) protein, and neutralizing antibodies during a period of six months following COVID-19 disease onset in 349 symptomatic COVID-19 patients, which were among the first world-wide being infected. The positivity rate and magnitude of IgM-S and IgG-N responses increased rapidly. High levels of IgM-S/N and IgG-S/N at 2-3 weeks after disease onset were associated with virus control and IgG-S titers correlated closely with the capacity to neutralize SARS-CoV-2. While specific IgM-S/N became undetectable 12 weeks after disease onset in most patients, IgG-S/N titers showed an intermediate contraction phase, but stabilized at relatively high levels over the six months observation period. At late time points the positivity rates for binding and neutralizing SARS-CoV-2-specific antibodies was still over 70%. Taken together, our data indicate sustained humoral immunity in recovered patients who suffer from symptomatic COVID-19, suggesting prolonged immunity.

Seasonality of non-SARS, non-MERS Corona viruses and the impact of meteorological factors (preprint)

BackgroundSeasonality is a characteristic of some respiratory viruses. The aim of our study was to evaluate the seasonality and the potential effects of different meteorological factors on the detection rate of the non-SARS Corona Virus detection by PCR. MethodsWe performed a retrospective analysis of 12763 respiratory tract sample results (288 positive and 12475 negative) for non-SARS, non-MERS Corona viruses (NL63, 229E, OC43, HKU1). The effect of seven single weather factors on the Corona virus detection rate was fitted in a logistic regression model with and without adjusting for other weather factors. ResultsCorona virus infections followed a seasonal pattern peaking from December to March and plunging from July to September. The seasonal effect was less pronounced in immunosuppressed patients compared to immunocompetent. Different automatic variable selection processes agreed to select the predictors temperature, relative humidity, cloud cover and precipitation as remaining predictors in the multivariable logistic regression model including all weather factors, with low ambient temperature, low relative humidity, high cloud cover and high precipitation being linked to increased Corona virus detection rates. ConclusionsCorona virus infections followed a seasonal pattern, which was more pronounced in immunocompetent patients compared to immunosuppressed. Several meteorological factors were associated with the Corona virus detection rate. However, when mutually adjusting for all weather factors, only temperature, relative humidity, precipitation and cloud cover contributed independently to predicting the Corona virus detection rate.

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.