Enabling mRNA medicine for brain tumors

mRNA is a new class of drugs that has the potential to revolutionize the treatment of brain tumors. Thanks to the COVID-19 mRNA vaccines and numerous therapy-based clinical trials, it is now clear that lipid nanoparticles (LNPs) are a clinically viable means to deliver RNA therapeutics. However, LNP-mediated mRNA delivery to brain tumors remains elusive. Over the past decade, numerous studies have shown that tumor cells communicate with each other via small extracellular vesicles, which are around 100 nm in diameter and consist of lipid bilayer membrane similar to synthetic lipidbased nanocarriers. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain tumors without undue toxicity. We synthesized LNPs using four components similar to the formulation used in the mRNA COVID19 vaccines (Moderna and Pfizer): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. For the in vitro screen, we tested ten classes of helper lipids based on their abundance in extracellular vesicle membranes, commercial availability, and large-scale production feasibility while keeping rest of the LNP components unchanged. The transfection kinetics of GFP mRNA encapsulated in LNPs and doped with 16 mol% of helper lipids was tested using GL261, U87 and SIM-A9 cell lines. Several LNP formations resulted in stable transfection (upto 5 days) of GFP mRNA in all the cell lines tested in vitro. The successful LNP candidates (enabling >80% transfection efficacy) were then tested in vivo to deliver luciferase mRNA to brain tumors via intrathecal administration in a syngeneic glioblastoma (GBM) mouse model, which confirmed luciferase expression in brain tumors in the cortex. LNPs were then tested to deliver Cre recombinase mRNA in syngeneic GBM mouse model genetically modified to express tdTomato under LoxP marker cassette that enabled identification of LNP targeted cells. mRNA was successfully delivered to tumor cells (70-80% transfected) and a range of different cells in the tumor microenvironment, including tumor-associated macrophages (80-90% transfected), neurons (31- 40% transfected), neural stem cells (39-62% transfected), oligodendrocytes (70-80% transfected) and astrocytes (44-76% transfected). Then, LNP formulations were assessed for delivering Cas9 mRNA and CD81 sgRNA (model protein) in murine syngeneic GBM model to enable gene editing in brain tumor cells. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~94% of brain tumor cells in vivo. In conclusion, we have developed a library of safe LNPs that can transfect GBM cells in vivo with high efficacy. This technology can potentially be used to develop novel mRNA therapies for GBM by delivering single or multiple mRNAs and holds great potential as a tool to study brain tumor biology.

Function and interaction of coronavirus ion channel proteins

Objective: COVID-19 has emerged as a significant global health crisis causing devastating effects on world population accounting for over 6 million deaths worldwide. Although acute RTI is the prevalent cause of morbidity, kidney outcomes centered on a spectrum of AKI have evolved over the course of the pandemic. Especially the emerging variants have posed a daunting challenge to the scientific communities, prompting an urging requirement for global contributions in understanding the viral dynamics. In addition to canonical genes, several subgroup- specific accessory genes are located between the S and E genes of coronaviruses regarding which little is known. Previous studies have shown that accessory proteins (aps) in viruses function as viroporins that regulate viral infection, propagation and egress [1]. In this study we attempted to characterize the function of aps of coronavirus variants as ion channels. Furthermore, we also probed the interaction of ap4 with the host system. Method(s): Serial passaging (selection pressure), growth kinetics, confocal imaging, genome sequence analysis and proteomics were performed in Huh-7, MRC5 cells and/or human monocyte derived macrophages. Potassium uptake assay was performed in a Saccharo myces cerevisiae strain, which lacks the potassium transporters trk1 and trk2. Ion conductivity experiments were performed in Xenopus laevis oocytes using Two Electrode Voltage Clamp (TEVC) method. Result(s): Serial passaging demonstrated the acquisition of several frameshift mutations in ORF4 resulting in C-terminally truncated protein versions (ap4 and ap4a) and indicate a strong selection pressure against retaining a complete ORF4 in vitro. Growth kinetics in primary cells illustrated a reduction of viral titers when the full-length ap4 was expressed compared to the C-terminally truncated protein ap4a. Confocal imaging showed that ap4 and ap4a are not exclusively located in a single cellular compartment. Potassium uptake assay in yeast and TEVC analyses in Xenopus oocytes showed that ap4 and ap4a act as a weak K+ selective ion channel. In addition, accessory proteins of other virus variants also elicited microampere range of currents. Conclusion(s): Our study provides the first evidence that ap4 and other accessory proteins of coronavirus variants act as viroporins. Future studies are aimed at demonstrating the role of ap4 during the viral life cycle by modulating ion homeostasis of host cell in vivo (interacting proteins obtained from proteomic studies) and thereby serve as a tool for potential drug target.

Kinetic Energy Recovery from a Landing Aircraft: Energy Analysis, Environmental and Economic Prospects

The Intergovernmental panel on Climate Change has sounded its alarm through its special report on the impact of global warming of 1.5oC and called for a strengthened global response to the threat of climate change. Despite that the COVID-19 pandemic has left a devastating effect on the aviation industry, this is forecasted to bounce back and recover within a few years. It is therefore important now to revisit opportunities for a better balance between social, environmental and economic impact of the sector. The European Union has been leading the way in limiting the environmental impacts of aviation. Despite that most of the R&D effort has been focused on the airborne phase, the European Union is legislating so that all aircraft movements on the ground are set to be emission-free by 2050. The paper focuses on engineless aircraft taxiing with the aim to reduce emissions on the ground. We demonstrate that upon landing, an aircraft has enough kinetic energy, which if recovered could power a 5-minute engineless taxiing process. When scaled to a large fleet such as low-cost carriers, this emissions problem can be turned on its head and becomes an opportunity for fuel savings and a reduction in emissions on the ground. The paper also demonstrates that the cost to retrofit such technology can be recovered in a short timeframe and therefore there is an economic incentive to the airline. © 2022 ICAS. All Rights Reserved.

A review on co-pyrolysis of agriculture biomass and disposable medical face mask waste for green fuel production: recent advances and thermo-kinetic models.

The Association of Southeast Asian Nations is blessed with agricultural resources, and with the growing population, it will continue to prosper, which follows the abundance of agricultural biomass. Lignocellulosic biomass attracted researchers' interest in extracting bio-oil from these wastes. However, the resulting bio-oil has low heating values and undesirable physical properties. Hence, co-pyrolysis with plastic or polymer wastes is adopted to improve the yield and quality of the bio-oil. Furthermore, with the spread of the novel coronavirus, the surge of single-use plastic waste such as disposable medical face mask, can potentially set back the previous plastic waste reduction measures. Therefore, studies of existing technologies and techniques are referred in exploring the potential of disposable medical face mask waste as a candidate for co-pyrolysis with biomass. Process parameters, utilisation of catalysts and technologies are key factors in improving and optimising the process to achieve commercial standard of liquid fuel. Catalytic co-pyrolysis involves a series of complex mechanisms, which cannot be explained using simple iso-conversional models. Hence, advanced conversional models are introduced, followed by the evolutionary models and predictive models, which can solve the non-linear catalytic co-pyrolysis reaction kinetics. The outlook and challenges for the topic are discussed in detail.

Within-host modeling to measure dynamics of antibody responses after natural infection or vaccination: A systematic review.

BACKGROUND: Within-host models describe the dynamics of immune cells when encountering a pathogen, and how these dynamics can lead to an individual-specific immune response. This systematic review aims to summarize which within-host methodology has been used to study and quantify antibody kinetics after infection or vaccination. In particular, we focus on data-driven and theory-driven mechanistic models. MATERIALS: PubMed and Web of Science databases were used to identify eligible papers published until May 2022. Eligible publications included those studying mathematical models that measure antibody kinetics as the primary outcome (ranging from phenomenological to mechanistic models). RESULTS: We identified 78 eligible publications, of which 8 relied on an Ordinary Differential Equations (ODEs)-based modelling approach to describe antibody kinetics after vaccination, and 12 studies used such models in the context of humoral immunity induced by natural infection. Mechanistic modeling studies were summarized in terms of type of study, sample size, measurements collected, antibody half-life, compartments and parameters included, inferential or analytical method, and model selection. CONCLUSIONS: Despite the importance of investigating antibody kinetics and underlying mechanisms of (waning of) the humoral immunity, few publications explicitly account for this in a mathematical model. In particular, most research focuses on phenomenological rather than mechanistic models. The limited information on the age groups or other risk factors that might impact antibody kinetics, as well as a lack of experimental or observational data remain important concerns regarding the interpretation of mathematical modeling results. We reviewed the similarities between the kinetics following vaccination and infection, emphasising that it may be worth translating some features from one setting to another. However, we also stress that some biological mechanisms need to be distinguished. We found that data-driven mechanistic models tend to be more simplistic, and theory-driven approaches lack representative data to validate model results.

The kinetics of SARS-CoV-2 viremia in COVID-19 patients receiving remdesivir.

Detection of SARS-CoV-2 RNA in serum, viremia, has been linked to disease severity and outcome. The kinetics of viremia in patients receiving remdesivir has not been thoroughly studied and could help predict treatment response and outcome. We investigated the kinetics of SARS-CoV-2 viremia and factors associated with baseline viremia, viral clearance and 30-day mortality in patients receiving remdesivir. An observational study including 378 hospitalised patients (median age 67 years, 67% male) sampled with serum SARS-CoV-2 RT-PCR within ± 24 h of initiation of remdesivir treatment. Baseline viremia was present in 206 (54%) patients with a median Ct value of 35.3 (IQR = 33.3-37.1). In patients with baseline viremia, the estimated probability of viral clearance was 72% by day 5. Ct values decreased significantly during remdesivir treatment for viremic patients, indicating an increase in viral load. In total, 44 patients (12%) died within 30 days, and mortality was significantly associated with viremia at baseline (OR = 2.45, p = 0.01) and lack of viral clearance by day 5 (OR = 4.8, p = < 0.01). Viral clearance was not associated with any individual risk factor. Viremia appears to be a prognostic marker before and during remedesivir treatment. The resolution of viremia was similar to patients not receiving remdesivir in other studies, and the decrease in Ct values during treatment questions the antiviral capacity of remdesivir in vivo. Prospective studies are warranted to confirm our findings.

Severe Acute Respiratory Syndrome Coronavirus 2 Infection History and Antibody Response to 3 Coronavirus Disease 2019 Messenger RNA Vaccine Doses.

BACKGROUND: Data on antibody kinetics are limited among individuals previously infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). From a cohort of healthcare personnel and other frontline workers in 6 US states, we assessed antibody waning after messenger RNA (mRNA) dose 2 and response to dose 3 according to SARS-CoV-2 infection history. METHODS: Participants submitted sera every 3 months, after SARS-CoV-2 infection, and after each mRNA vaccine dose. Sera were tested for antibodies and reported as area under the serial dilution curve (AUC). Changes in AUC values over time were compared using a linear mixed model. RESULTS: Analysis included 388 participants who received dose 3 by November 2021. There were 3 comparison groups: vaccine only with no known prior SARS-CoV-2 infection (n = 224); infection prior to dose 1 (n = 123); and infection after dose 2 and before dose 3 (n = 41). The interval from dose 2 and dose 3 was approximately 8 months. After dose 3, antibody levels rose 2.5-fold (95% confidence interval [CI] = 2.2-3.0) in group 2 and 2.9-fold (95% CI = 2.6-3.3) in group 1. Those infected within 90 days before dose 3 (and median 233 days [interquartile range, 213-246] after dose 2) did not increase significantly after dose 3. CONCLUSIONS: A third dose of mRNA vaccine typically elicited a robust humoral immune response among those with primary vaccination regardless of SARS-CoV-2 infection >3 months prior to boosting. Those with infection <3 months prior to boosting did not have a significant increase in antibody concentrations in response to a booster.

The Kinetics of Anti-SARS-CoV-2 Antibodies in Pediatric Patients and the Characterization of Post-COVID-19 Condition at 6 Months After Infection: Protocol for a Longitudinal Observational Study.

BACKGROUND: Data regarding the kinetics of anti-SARS-CoV-2 antibodies and information about post-COVID-19 condition (colloquially known as "long COVID") in children are scarce, especially in low-income countries. Even though cases of COVID-19 in children are less prevalent than adults, post-COVID-19 condition cases in children are high and have a burden that may impact their growth and development. There are other features of antibody kinetics in connection with SARS-CoV-2 infection that are yet unknown as of this writing, especially in children following infection. Furthermore, the long-term results, risk factors, and underlying pathophysiology are still uncertain. To better understand post-COVID-19 condition in children, it is necessary to further investigate the impact of clinically significant factors such multisystem inflammatory syndrome and disease severity among hospitalized survivors through their SARS-CoV-2 antibody response. OBJECTIVE: We aim to analyze anti-receptor-binding domain SARS-CoV-2 immunoglobulin G antibodies over time and characterize the signs and symptoms of post-COVID-19 condition in pediatric patients at the time of diagnosis and at 2 weeks and 1, 3, and 6 months following infection. METHODS: This is a longitudinal observational study in Indonesia. Pediatric patients diagnosed with COVID-19 by positive molecular assay using nasopharyngeal swab will be tested for anti-SARS-CoV-2 antibodies using the Roche Elecsys Anti-SARS-CoV-2 S assay at the time of diagnosis and at 2 weeks and 1, 3, and 6 months following infection. Antibody titer data will be reported as means and SDs. The respondents' signs and symptoms will be observed up to 6 months after the onset of infection, including the vaccination event, reinfection, rehospitalization, and mortality. The clinical features will be reported as frequencies and percentages. RESULTS: Participant enrollment began in February 2022. As of September 30, 2022, a total of 58 patients were enrolled. After data collection, results are expected to be analyzed in August 2023. CONCLUSIONS: This study will allow us to know the kinetics of anti-receptor-binding domain SARS-CoV-2 immunoglobulin G antibodies and data regarding post-COVID-19 condition up to 6 months following infection in the Indonesian pediatric population. Furthermore, this study has the potential to serve as a foundation for government decisions about vaccination programs and prevention measures. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/43344.

Immunological Response to Conventional COVID-19 Vaccines in a Cohort of Pakistani Healthy Recipients

Coronavirus disease 2019 mass vaccination has led to drastic reduction in hospitalizations and mortality. A number of case reports have emerged reporting coronavirus disease 2019 infection within days following vaccination. There is a need to understand development of immune antibodies in the early post-vaccination period. A prospective analysis of immunoglobulin M and immunoglobulin G kinetics was conducted during the first 28 d following vaccination with either CanSino or Sinovac vaccines in a cohort of 40 healthy volunteers. Serial blood samples were collected from the volunteers right before the first dose of vaccine (d 0) and then on d 4, d 7, d 14, d 21, d 24 and d 28 post-vaccination. Using enzyme-linked immunosorbent assay, circulating anti-severe acute respiratory syndrome coronavirus 2 receptor binding domain immunoglobulin M and immunoglobulin G antibodies were analyzed. Most vaccine recipients (31/40) did not develop any circulating immunoglobulin M. The remaining 9 recipients showed a typical immunoglobulin M curve with antibodies appearing on d 4, peaking on d 7 and declining on d 21 and beyond. Immunoglobulin G response was more typical within 38/40 recipients showing the appearance of immunoglobulin G on d 4, which continued till the end of the study period. This study demonstrates that vaccine-induced immunoglobulin M-based immunity cannot be relied during the first few days following vaccination and more time is needed to have a better picture of the real situation.

Kinetics of Anti-Sars-Cov-2 Igg Antibodies Post-Covid-19 -Vaccination

Intro: During the pandemic of COVD-19, several vaccines have been developed and are currently used worldwide. However, head-to-head studies comparing various vaccines are limited. Therefore, This single-center, prospective, realworld study. head-to-head study of compared the effectiveness of BNT162b2 (Pfizer BioNTech), mRNA-1273 (Moderna) ChAdOx1-S (Astra Zeneca) vaccines. The kinetics of SARS-CoV2 spike antibodies were monitored post-vaccination and following two booster doses in COVID-19 naive and previously infected adults. Method(s): The primary outcome was the emergence of virologically positive COVID-19 cases after vaccine completion. The secondary outcome was the occurrence of postvaccination COVID-19-related hospitalizations. The titers of anti-SARS-CoV-2 IgG antibodies against the S1 subunit of the virus's spike protein were measured after the first and second doses of the three vaccines and the booster doses. Finding(s): The current study enrolled and followed 1550 participants who received ChAdOx1-S or BNT162b2, or mRNA-1273, and 1550 non-vaccinated subjects between March 2021 and February 2022. After completing two vaccine doses, the effectiveness in preventing COVID-19 cases was 89.2%, 95.5%, and 94.6% for ChAdOx1-S or BNT162b2, or mRNA-1273, respectively. Four COVID-19-related hospitalizations (0.26%) were reported in the vaccinated versus 648 (41.81%) non-vaccinated participants (P<0.0001). Following the Pfizer booster dose, no COVID-19-positive cases or hospitalizations were reported. In the three vaccines, SARS-CoV2 antibody titers increased gradually after the first dose, peaked 3-4 weeks after the second dose, then declined after 28-32 weeks. Enhanced antibody response was observed after the booster dose and was maintained until the end of follow-up. Comorbidities were associated with lower antibody titers, particularly diabetes, autoimmune diseases, and advanced renal diseases. Conclusion(s): The study showed that the three COVID-10 vaccines effectively reduced the risk of virologically confirmed COVID-19 disease and prevented severe illness and hospitalizations in COVID-19 naiive and previously infected. Booster doses enhance the SARS-CoV2 antibody response and decrease the incidence of virologically proven severe COVID-19.Copyright © 2023

Optimization of Thrombinemia Laboratory Diagnostics in COVID-19-Patients

Introduction. Currently used and available in real clinical practice, laboratory tests do not allow an objective and reliable assessment of risk and severity of thrombinemia, prothrombotic readiness, and, as a result, does not allow to choose the appropriate optimal antithrombotic therapy regimen (administration of prophylactic or therapeutic anticoagulant doses) in relation to the screening of this condition in severe COVID-19. Objective: analysis of prothrombotic readiness in COVID-19-patients by integral method — thrombin generation (kinetics) assay. Materials and Methods. A prospective clinical and laboratory study included 100 patients (average age was 63 [31–85] years, 60 women, 40 men) with an identified SARS-CoV-2 virus with moderate and severe course of novel coronovirus infection. Parameters of thrombin generation (kinetics) assay — clotting initiation time (tLag), time of thrombin peak formation (tPeak), thrombin peak (Peak), endogenous thrombin potential (ETR;area under the thrombin formation curve, AUC) were determined, as well as levels of fibrinogen, D-dimer, ferritin, C-reactive protein (CRP), activated partial thromboplastin time (APTT), prothrombin time (PT), international normalized ratio (INR). Results. The results of thrombin kinetics assay pointed to an increased blood procoagulant potential in COVID-19-patients on admission to the hospital;correlations were found between tLag and fibrinogen (rS = –0.7;p = 0.001), tLag and CRP (rS = –0.1;p = 0.01);between tPeak and all inflammation markers — D-dimer (rS = –0.4;p = 0.001), ferritin (rS = –0.2;p = 0.01), CRP (rS = –0.3;p = 0.05), fibrinogen (rS = –0.5;p = 0.001);between Peak and fibrinogen (rS = 0.3;p = 0.001);between rate index (VI) and fibrinogen (rS = 0.2;p < 0.001);between AUC and fibrinogen (rS = 0.4;p = 0.001), AUC and CRP (rS = 0.1;p = 0.001). Conclusion. Thrombin generation (kinetics) assay can be considered as a marker of thrombinemia — prothrombotic readiness in patients with moderate and severe course of novel coronovirus infection. © 2023, Hemostasis and Rheology LLC. All rights reserved.

Forecasting COVID-19 cases in Egypt using ARIMA-based time-series analysis

Objectives: The World Health Organization declared the novel coronavirus (COVID-19) outbreak a public health emer-gency of international concern on January 30, 2020. Since it was first identified, COVID-19 has infected more than one hundred million people worldwide, with more than two million fatalities. This study focuses on the interpretation of the distribution of COVID-19 in Egypt to develop an effective forecasting model that can be used as a decision-making mechanism to administer health interventions and mitigate the transmission of COVID-19. Method(s): A model was developed using the data collected by the Egyptian Ministry of Health and used it to predict possible COVID-19 cases in Egypt. Result(s): Statistics obtained based on time-series and kinetic model analyses suggest that the total number of CO-VID-19 cases in mainland Egypt could reach 11076 per week (March 1, 2020 through January 24, 2021) and the number of simple regenerations could reach 12. Analysis of the ARIMA (2, 1, 2) and (2, 1, 3) sequences shows a rise in the number of COVID-19 events. Conclusion(s): The developed forecasting model can help the government and medical personnel plan for the imminent conditions and ensure that healthcare systems are prepared to deal with them.Copyright © 2021 by Eurasian Journal of Medicine and Oncology.

Unbalanced networks and disturbed kinetics of serum soluble mediators associated with distinct disease outcomes in severe COVID-19 patients.

The present study applied distinct models of descriptive analysis to explore the integrative networks and the kinetic timeline of serum soluble mediators to select a set of systemic biomarkers applicable for the clinical management of COVID-19 patients. For this purpose, a total of 246 participants (82 COVID-19 and 164 healthy controls - HC) were enrolled in a prospective observational study. Serum soluble mediators were quantified by high-throughput microbeads array on hospital admission (D0) and at consecutive timepoints (D1-6 and D7-20). The results reinforce that the COVID-19 group exhibited a massive storm of serum soluble mediators. While increased levels of CCL3 and G-CSF were associated with the favorable prognosis of non-mechanical ventilation (nMV) or discharge, high levels of CXCL10 and IL-6 were observed in patients progressing to mechanical ventilation (MV) or death. At the time of admission, COVID-19 patients presented a complex and robust serum soluble mediator network, with a higher number of strong correlations involving IFN-γ, IL-1Ra and IL-9 observed in patients progressing to MV or death. Multivariate regression analysis demonstrates the ability of serum soluble mediators to cluster COVID-19 from HC. Ascendant fold change signatures and the kinetic timeline analysis further confirmed that the pairs "CCL3 and G-CSF" and "CXCL10 and IL-6" were associated with favorable or poor prognosis, respectively. A selected set of systemic mediators (IL-6, IFN-γ, IL-1Ra, IL-13, PDGF and IL-7) were identified as putative laboratory markers, applicable as complementary records for the clinical management of patients with severe COVID-19.

Inhibition of the SARS-CoV-2 Main Protease by Isoquercitrin γ-Cyclodextrin Inclusion Complex Formulations: A Biochemical and In Silico Study

The main protease (Mpro) of SARS-CoV-2 is a well-established drug target for rational drug design of COVID-19 inhibitors. To address the serious challenge of COVID-19, we have performed biochemical inhibition screens with recombinantly expressed SARS-CoV-2 main protease (Mpro). A fluorescent assay was used to identify the flavonoid isoquercitrin as an Mpro inhibitor. Both isoquercitrin encapsulated in γ-cyclodextrin (inclusion complex formulations) and alone inhibited SARS-CoV-2 Mpro. For isoquercitrin, a Ki value of 32 μM (IC50 = 63 μM) was obtained. Isoquercitrin γ-cyclodextrin inclusion complex formulations additionally inhibited Zika virus NS2B-NS3pro leading to an IC50 value of 98 μM. Formulations containing the other flavonoid compounds diosmetin-7-O-glucoside, hesperetin-7-O-glucoside, and naringenin-7-O-glucoside did not inhibit SARS-CoV-2 Mpro. Steady-state kinetics indicate that the inhibition mechanism of Mpro by isoquercitrin is potentially competitive. Molecular modeling studies carried out with MM/PBSA confirm the likely modes of isoquercitrin binding to both proteases. These modeling results can be used in the development of structural analogs of isoquercitrin with better inhibitory profiles and potential candidates for anti-coronavirus drugs. Since the targeted proteases are essential for viral activity, the delivery isoquercitrin-cyclodextrin inclusion complex formulations could be of great interest for the development of future antiviral drugs to target intracellular virus proteins or other components.

SARS-CoV-2 BA.5 CAUSES HIGHER REPLICATION AND LETHAL INFECTION IN K18-hACE2 MICE

Background: The continuous evolution of SARS-CoV-2 in the diverse immune landscape (natural, vaccine, hybrid) is giving rise to novel immune escape mutations. So far, the resulting new variants (BA.1, BA.2, BA.2.12.1) were observed to cause mild infections, however, BA.5 infections are associated with an increased risk of hospitalization.1 Therefore it is essential to investigate the pathogenesis of BA.5. Method(s): Here we compared the pathogenicity of Pre-Omicron (B.1.351) and Omicron (BA.1, BA.2.12.1, and BA.5) variants in wild-type C57BL/6J mice and K18-hACE2 mice. The virus replication kinetics was also studied in human Calu3, pulmonary alveolar type 2 (AT2) cells, and airway organoids (HAO). Cell-to-cell spread of virus was measured by syncytia formation assay and immunohistochemistry (IHC) of infected lungs. Result(s): In the results, infection in C57BL/6J mice showed severe weight loss ( >15%) for B.1.351 infected mice and moderate ( >5%) for BA.5 infected. C57BL/6J mice showed higher virus replication of B.1.351 followed by BA.5, BA.1, and BA.2.12.1. At the peak of virus replication (2 days) plaque-forming units from lung extract of BA.5 infected mice were two, and three logs higher compared to BA.1 and BA.2.12.1 respectively. BA.5 infection was lethal to 80% of infected K18-hACE2 mice, whereas the mice looked normal after infection with BA.1 and BA.2.12.1. BA.5 infected mice showed high virus replication in brain tissue. Surprisingly the syncytia formation assay and IHC for BA.5 was comparable to that of B.1.351, indicating the higher cell-to-cell spread of BA.5 and B.1.351 compared to BA.1 and BA.2.12.1, which is one of the measures of pathogenicity. Calu3 and HAO showed the same trend of virus replication as was observed in-vivo experiments however AT2 cells were found to be resistant to BA.5 replication. Conclusion(s): These results suggest that the BA.5 variant (lineage) of Omicron has the potential to regain the pathogenicity as it shows increased virulence compared to other Omicron sub-variants. Lethal infection of BA.5 in K18-hACE2 mice may be attributed to catastrophic encephalitis and increased cell-to-cell spread.

Tolerance of ETD001, a long-acting inhaled epithelial sodium channel blocker, in humans

Background: To assess the safety, tolerability, and pharmacokinetic (PK) profile in humans of the novel inhaled epithelial sodium channel blocker ETD001. Method(s): Inhaled ETD001 or placebo, delivered via nebulizer, have been administered in a 3:1 ratio to 96 healthy subjects in a blinded, first-inhuman clinical trial (ClinicalTrials.gov Identifier: NCT04926701). The study consisted of two parts. Part A evaluated single ascending doses (SADs) up to 10.8 mg, and Part B evaluated multiple ascending doses (MADs) up to 3.1 mg once daily (QD) for 7 days and 4.65 mg twice daily (BID) for 14 days. Safety was assessed by monitoring for adverse events (AEs), laboratory safety tests (including blood potassium monitoring), vital signs, 12-lead electrocardiogram (ECG), and spirometry. Systemic exposurewas assessed using serial pharmacokinetic blood draws. Result(s): Therewere no serious AEs. Twenty-four subjects reported 38 AEs, all of mild to moderate intensity and all resolved. There were no clinically relevant changes in laboratory safety tests, vital signs, ECGs, or spirometry measurements. All blood potassium assessments were within normal range at all doses. Three subjects withdrew in Part B;all withdrawals were considered unrelated to study drug: one on day 6 from the 3.1-mg QD cohort for personal reasons, one after the first dose of the 3.1-mg BID cohort because of vasovagal syncope at time of venipuncture triggering atrial fibrillation that spontaneously resolved, and one on Day 4 of the 3.1- mg BID cohort because of a positive COVID-19 test. Pharmacokinetic parameters were approximately dose proportional in Part A, with peak concentrations 1 to 2 hours after dose and exposure out to 12 to 24 hours at all doses, indicating good lung retention. Part B plasma concentrations displayed dose-independent kinetics and showed minimal accumulation, with a mean of 1.11-fold observed over 14 days. Conclusion(s): ETD001 was well tolerated at single doses up to 10.8 mg and multiple doses of 3.1 mg QD for 7 days and 4.65 mg BID for 14 days. The wide safety margin is predicted to enable doses capable of durable target engagement in the lung, which are expected to enhance mucociliary clearance in people with cystic fibrosis.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Viral Dynamics and Factors Favouring the Duration of Covid-19 Positivity in Cameroon

Background: Evolution evidence of Coronavirus disease 2019 (COVID-19) and viral clearance time remains limited in tropical settings. Understanding this is crucial for public health control measures at community-level. We evaluated the viral dynamics of SARS-CoV-2 infection and factors associated with positivity duration in COVID-19 cases in Cameroon. Method(s): We conducted a prospective cohort-study of SARS-CoV-2 positive cases from the first to third wave (March 2020-October 2021) in Yaounde- Cameroon. RT-PCR was performed on nasopharyngeal swabs. SARS-CoV-2 positivity duration was evaluated from the first to last positive test before a negative result. Epi-info V.7.0 was used for data analyses with p< 0.05 considered statistically significant Results: A total of 282 participants were enrolled. The mean age was 41+/-14 years, with male predominant (62.1%). We had 15.6% symptomatic cases and cough most common (59.09%). The overall median positivity duration was 15[IQR: 9-23] days with 15[ IQR: 13-16] in the first, 17[ IQR: 11-26] in the second and 8[ IQR: 4-12] in the third wave (p= 0.007). Positivity duration was significantly higher in males (16 versus 14 days, p=0.03) and people aged >40 years (15 versus 14 days, p=0.02). Positivity duration was not affected by presence or absence of symptoms (p=0.80). No significant correlation was found with viral load (r=0.03;p=0.61). Considering baseline (24.7+/-7.2Ct) and last viral load (29.3+/-5.9 Ct), the DELTACt (4.6+/-1.3) and positivity duration (15 days) revealed a kinetic in viral decay of 0.3+/-0.087 Ct/day. Conclusion(s): A median positivity duration of 15 days is in accordance with viral clearance around 2 weeks for optimal confinement at community-level. Men and/or the elderly stand at higher risk of prolonged infection. Given the viral decay (0.3 Ct daily), we suggest personalized confinement periods. The variability of positivity duration according to phases could be function of strains which could be a factor of positivity duration.

A Randomized Trial on Early Therapy in Covid-19 High-Risk Outpatients in Omicron Era

Background: Antivirals and monoclonal antibodies (mAbs) were approved for early treatment of COVID-19 based on data from trials conducted in unvaccinated people before the Omicron era. The comparative effectiveness of different treatments is unknown. We present the results of the interim analysis of MONET trial (EudraCT: 2021-004188-28). Method(s): In this ongoing multicenter, open-label, phase 4 trial, we randomly assigned, in a 1:1:1 ratio, non-hospitalized patients with early symptomatic Covid-19 (<=5 days after symptoms onset) and >=1 risk factor for disease progression, to receive 500 mg of intravenous sotrovimab (SOT) or 600 mg of intramuscular tixagevimab/cilgavimab (TIX/CIL) or oral 5-days course of NMV/r 300/100 mg BID. Primary outcome was hospitalization or death for any cause within 29 days after randomization, reported as cumulative incidence per 100 (95% CI), and P-value calculated by Fisher's exact test. Inflammatory marker (CRP, d-dimer, and neutrophils-to-lymphocytes ratio) and antibody level (serum anti-S IgG and anti-N IgG) analysed by mixed linear regression with random intercept and P-values for time trend calculated by ANOVA-style test with Bonferroni correction. Result(s): Prespecified interim analysis, including 400 patients (SOT =133, TIX/ CIL=130, NMV/r=137) enrolled from Mar 4 to Nov 16, 2022 (Fig.1A). Overall, 5 pts (3/5 immunosuppressed) had disease progression leading to hospitalization [1.25% (95% CI 0.4%-2.89%)], 1 in SOT (0.75%, 95% CI 0.01%-4.1%), 4 in TIX/CIL (3.08%, 95% CI 0.84%-7.69%) and none in NMV/r arm (P=0.030). No deaths or ICU admissions were observed. Among the hospitalized pts, 3 were infected with BA.2 (1 SOT, 2 TIX/CIL), one with BA.4/5, and one BQ.1.1 (both TIX/ CIL). No serious adverse events and no kidney or liver toxicity were reported. Temporal trend of inflammation markers was similar in the three arms, and their estimates are shown in Fig.1B. Kinetics of antibody was reported in Fig.1C. The plot shows a rapid increase of anti-S in both mAb arm and a linear increase of IgG in the NMV/r arm. Anti-N IgG kinetics was similar in the three arms. Conclusion(s): By these data the overall cumulative risk of clinical failure in mild Covid-19 occurring in the Omicron era is low. The hypothesis that differences in clinical progression among the three arms could be related to different activity against the Omicron subvariant observed in vitro should be further investigated. Type of treatment does not seem to influence the development of the natural antibody response.

Population pharmacokinetics of dexamethasone in critically ill COVID-19 patients: inflammation might play a role

Introduction: One of the common causes of COVID-19 related death is acute respiratory distress syndrome (C-ARDS). Dexamethasone is the cornerstone in the therapy of C-ARDS and reduces mortality probably by suppressing inflammatory levels in ICU patients. Its anti-inflammatory effects may be concentration-related. However, no pharmacokinetic studies of dexamethasone have been conducted in ICU patients. Therefore, we designed a population pharmacokinetic study to gain a deeper understanding of the pharmacokinetics of dexamethasone in critically ill patients in order to identify relevant covariates that can be used to personalize dosing regimens and improve clinical outcomes. Method(s): This was a retrospective pilot study at the ICU of the Erasmus Medical Center. Blood samples were collected in adults at the ICU with COVID who were treated with fixed dose intravenous dexamethasone (6 mg/day). The data were analyzed using Nonlinear Mixed Effects Modelling (NONMEM) software for population pharmacokinetic analysis and clinically relevant covariates were selected and evaluated. Result(s): A total of 51 dexamethasone samples were measured in 18 patients. A two-compartment model with first-order kinetics best fitted the data. The mean population estimates for drug clearance and inter-compartment clearance were 2.85 L/h (IIV 62.9%) and 2.11 L/h, respectively, and central and peripheral volumes of distribution were 15.4 L and 12.3 L, respectively. The covariate analysis showed a significant correlation between dexamethasone clearance and CRP. Dexamethasone clearance decreased significantly with increasing CRP in the range of 0-50 mg/L and a correlation was observed with CRP up to 100 mg/L. Conclusion(s): The dexamethasone PK parameters of ICU COVID patients were quite different from those come from healthy populations. Inflammation might play an important role in dexamethasone clearance and the dosing should be more individualized in order to achieve best therapeutic effect in ICU patients.