On-target versus off-target effects of drugs inhibiting the replication of SARS-CoV-2

The current epidemic of coronavirus disease-19 (COVID-19) caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for the development of inhibitors of viral replication. Here, we performed a bioinformatic analysis of published and purported SARS-CoV-2 antivirals including imatinib mesylate that we found to suppress SARS-CoV-2 replication on Vero E6 cells and that, according to the published literature on other coronaviruses is likely to act on-target, as a tyrosine kinase inhibitor. We identified a cluster of SARS-CoV-2 antivirals with characteristics of lysosomotropic agents, meaning that they are lipophilic weak bases capable of penetrating into cells. These agents include cepharentine, chloroquine, chlorpromazine, clemastine, cloperastine, emetine, hydroxychloroquine, haloperidol, ML240, PB28, ponatinib, siramesine, and zotatifin (eFT226) all of which are likely to inhibit SARS-CoV-2 replication by non-specific (off-target) effects, meaning that they probably do not act on their ‘official’ pharmacological targets, but rather interfere with viral replication through non-specific effects on acidophilic organelles including autophagosomes, endosomes, and lysosomes. Imatinib mesylate did not fall into this cluster. In conclusion, we propose a tentative classification of SARS-CoV-2 antivirals into specific (on-target) versus non-specific (off-target) agents based on their physicochemical characteristics

Evidences for lipid involvement in SARS-CoV-2 cytopathogenesis

The pathogenesis of SARS-CoV-2 remains to be completely understood, and detailed SARS-CoV-2 cellular cytopathic effects requires definition. We performed a comparative ultrastructural study of SARS-CoV-1 and SARS-CoV-2 infection in Vero E6 cells and in lungs from deceased COVID-19 patients. SARS-CoV-2 induces rapid death associated with profound ultrastructural changes in Vero cells. Type II pneumocytes in lung tissue showed prominent altered features with numerous vacuoles and swollen mitochondria with presence of abundant lipid droplets. The accumulation of lipids was the most striking finding we observed in SARS-CoV-2 infected cells, both in vitro and in the lungs of patients, suggesting that lipids can be involved in SARS-CoV-2 pathogenesis. Considering that in most cases, COVID-19 patients show alteration of blood cholesterol and lipoprotein homeostasis, our findings highlight a peculiar important topic that can suggest new approaches for pharmacological treatment to contrast the pathogenicity of SARS-CoV-2

Human Zika infection induces a reduction of IFN-γ producing CD4 T-cells and a parallel expansion of effector Vδ2 T-cells

The definition of the immunological response to Zika (ZIKV) infection in humans represents a key issue to identify protective profile useful for vaccine development and for pathogenesis studies. No data are available on the cellular immune response in the acute phase of human ZIKV infection, and its role in the protection and/or pathogenesis needs to be clarified. We studied and compared the phenotype and functionality of T-cells in patients with acute ZIKV and Dengue viral (DENV) infections. A significant activation of T-cells was observed during both ZIKV and DENV infections. ZIKV infection was characterized by a CD4 T cell differentiation toward effector cells and by a lower frequency of IFN-γ producing CD4 T cells. Moreover, a substantial expansion of CD3+CD4−CD8− T-cell subset expressing Vδ2 TCR was specifically observed in ZIKV patients. Vδ2 T cells presented a terminally differentiated profile, expressed granzyme B and maintained their ability to produce IFN-γ. These findings provide new knowledge on the immune response profile during self-limited infection that may help in vaccine efficacy definition, and in identifying possible immuno-pathogenetic mechanisms of severe infection

Detection of Viral RNA in Tissues following Plasma Clearance from an Ebola Virus Infected Patient

An unprecedented Ebola virus (EBOV) epidemic occurred in 2013–2016 in West Africa. Over this time the epidemic exponentially grew and moved to Europe and North America, with several imported cases and many Health Care Workers (HCW) infected. Better understanding of EBOV infection patterns in different body compartments is mandatory to develop new countermeasures, as well as to fully comprehend the pathways of human-to-human transmission. We have longitudinally explored the persistence of EBOV-specific negative sense genomic RNA (neg-RNA) and the presence of positive sense RNA (pos-RNA), including both replication intermediate (antigenomic-RNA) and messenger RNA (mRNA) molecules, in the upper and lower respiratory tract, as compared to plasma, in a HCW infected with EBOV in Sierra Leone, who was hospitalized in the high isolation facility of the National Institute for Infectious Diseases “Lazzaro Spallanzani” (INMI), Rome, Italy. We observed persistence of pos-RNA and neg-RNAs in longitudinally collected specimens of the lower respiratory tract, even after viral clearance from plasma, suggesting possible local replication. The purpose of the present study is to enhance the knowledge on the biological features of EBOV that can contribute to the human-to-human transmissibility and to develop effective intervention strategies. However, further investigation is needed in order to better understand the clinical meaning of viral replication and shedding in the respiratory tract

Emulation of a Target Trial From Observational Data to Compare Effectiveness of Casirivimab/Imdevimab and Bamlanivimab/Etesevimab for Early Treatment of Non-Hospitalized Patients With COVID-19

OBJECTIVES: Comparative analysis between different monoclonal antibodies (mAbs) against SARS-CoV-2 are lacking. We present an emulation trial from observational data to compare effectiveness of Bamlanivimab/Etesevimab (BAM/ETE) and Casirivimab/Imdevimab (CAS/IMD) in outpatients with early mild-to-moderate COVID-19 in a real-world scenario of variants of concern (VoCs) from Alpha to Delta. METHODS: Allocation to treatment was subject to mAbs availability, and the measured factors were not used to determine which combination to use. Patients were followed through day 30. Viral load was measured by cycle threshold (CT) on D1 (baseline) and D7. Primary outcome was time to COVID-19-related hospitalization or death from any cause over days 0-30. Weighted pooled logistic regression and marginal structural Cox model by inverse probability weights were used to compare BAM/ETE vs. CAS/IMD. ANCOVA was used to compare mean D7 CT values by intervention. Models were adjusted for calendar month, MASS score and VoCs. We evaluated effect measure modification by VoCs, vaccination, D1 CT levels and enrolment period. RESULTS: COVID19-related hospitalization or death from any cause occurred in 15 of 237 patients in the BAM/ETE group (6.3%) and in 4 of 196 patients in the CAS/IMD group (2.0%) (relative risk reduction [1 minus the relative risk] 72%; p=0.024). Subset analysis carried no evidence that the effect of the intervention was different across stratification factors. There was no evidence in viral load reduction from baseline through day 7 across the two groups (+0.17, 95% -1.41;+1.74, p=0.83). Among patients who experienced primary outcome, none showed a negative RT-PCR test in nasopharyngeal swab (p=0.009) and 82.4% showed still high viral load (p<0.001) on D7. CONCLUSIONS: In a pre-Omicron epidemiologic scenario, CAS/IMD reduced risk of clinical progression of COVID-19 compared to BAM/ETE. This effect was not associated with a concomitant difference in virological response

Retrospective screening of solid organ donors in Italy, 2009, reveals unpredicted circulation of West Nile virus

Since the occurrence of West Nile virus (WNV) infection in humans in 2008 in Italy, concerns have been raised about the potential risks associated with solid organ transplantation (SOT). A nationwide retrospective survey showed that 1.2% of SOT donors in 2009 were WNV-seropositive and demonstrated that human WNV infection is distributed throughout several Italian regions. Transmission of WNV or other arboviruses through SOT is a possibility and risk assessment should be carried out before SOT to avoid infection through transplantatation

Frequency and duration of SARS-CoV-2 shedding in oral fluid samples assessed by a modified commercial rapid molecular assay

Background: RT-PCR on nasopharyngeal (NPS)/oropharyngeal swabs is the gold standard for diagnosis of SARS-CoV-2 infection and viral load monitoring. Oral fluid (OF) is an alternate clinical sample, easy and safer to collect and could be useful for COVID-19 diagnosis, monitoring viral load and shedding. Methods: Optimal assay conditions and analytical sensitivity were established for the commercial Simplexa™ COVID-19 Direct assay adapted to OF matrix. The assay was used to test 337 OF and NPS specimens collected in parallel from 164 hospitalized patients; 50 bronchoalveolar lavage (BAL) specimens from a subgroup of severe COVID-19 cases were also analysed. Results: Using Simplexa™ COVID-19 Direct on OF matrix, 100% analytical detection down to 1 TCID50/mL (corresponding to 4 × 103 copies (cp)/mL) was observed. No crossreaction with other viruses transmitted through the respiratory toute was observed. Parallel testing of 337 OF and NPS samples showed highly concordant results (κ = 0.831; 95 % CI = 0.771–0.891), and high correlation of Ct values (r = 0.921; p &lt; 0.0001). High concordance and elevated correlation was observed also between OF and BAL. Prolonged viral RNA shedding was observed up to 100 days from symptoms onset (DSO), with 32% and 29% positivity observed in OF and NPS samples, respectively, collected between 60 and 100 DSO. Conclusions: Simplexa™ COVID-19 Direct assays on OF have high sensitivity and specificity to detect SARS-CoV-2 RNA and provide an alternative to NPS for diagnosis and monitoring SARS-CoV-2 shedding