Enhancer deregulation inTET2-mutant clonal hematopoiesis is associated with increased COVID-19 severity and mortality

Background: COVID-19 causes significant morbidity and mortality, albeit with considerable heterogeneity among affected individuals. It remains unclear which host factors determine disease severity and survival. Given the propensity of clonal hematopoiesis (CH) to promote inflammation in healthy individuals, we investigated its effect on COVID-19 outcomes. Method(s): We performed a multi-omics interrogation of the genome, epigenome, transcriptome, and proteome of peripheral blood mononuclear cells from COVID-19 patients (n=227). We obtained clinical data, laboratory studies, and survival outcomes. We determined CH status and TET2-related DNA methylation. We performed single-cell proteogenomics to understand clonal composition in relation to cell phenotype. We interrogated single-cell gene expression in isolation and in conjunction with DNA accessibility. We integrated these multi-omics data to understand the effect of CH on clonal composition, gene expression, methylation of cis-regulatory elements, and lineage commitment in COVID-19 patients. We performed shRNA knockdowns to validate the effect of one candidate transcription factor in myeloid cell lines. Result(s): The presence of CH was strongly associated with COVID-19 severity and all-cause mortality, independent of age (HR 3.48, 95% CI 1.45-8.36, p=0.005). Differential methylation of promoters and enhancers was prevalent in TET2-mutant, but not DNMT3A-mutant CH. TET2- mutant CH was associated with enhanced classical/intermediate monocytosis and single-cell proteogenomics confirmed an enrichment of TET2 mutations in these cell types. We identified celltype specific gene expression changes associated with TET2 mutations in 102,072 single cells (n=34). Single-cell RNA-seq confirmed the skewing of hematopoiesis towards classical and intermediate monocytes and demonstrated the downregulation of EGR1 (a transcription factor important for monocyte differentiation) along with up-regulation of the lncRNA MALAT1 in monocytes. Combined scRNA-/scATAC-seq in 43,160 single cells (n=18) confirmed the skewing of hematopoiesis and up-regulation of MALAT1 in monocytes along with decreased accessibility of EGR1 motifs in known cis-regulatory elements. Using myeloid cell lines for functional validation, shRNA knockdowns of EGR1 confirmed the up-regulation of MALAT1 (in comparison to wildtype controls). Conclusion(s): CH is an independent prognostic factor in COVID-19 and skews hematopoiesis towards monocytosis. TET2-mutant CH is characterized by differential methylation and accessibility of enhancers binding myeloid transcriptions factors including EGR1. The ensuing loss of EGR1 expression in monocytes causes MALAT1 overexpression, a factor known to promote monocyte differentiation and inflammation. These data provide a mechanistic insight to the adverse prognostic impact of CH in COVID-19.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.

COVID-19 vaccination in pediatric age-what do parents know and think?

Background: Vaccination against COVID-19 in pediatric age has been a hot topic recently. This study aimed to assess parents' intention to vaccinate their children against COVID-19 and identify which factors may influence this decision. Methods: An observational study was conducted between June and July 2021 by applying an anonymous questionnaire to a sample of caregivers of children and adolescents followed in ambulatory care in a Portuguese central hospital. We included sociodemographic data, immunization history, personal background, exposure to COVID-19 and caregivers' beliefs. At the time of the study, there was still no official recommendation from health authorities regarding vaccination in pediatric age. Results: A total of 78 questionnaires were conducted. The mean age of the children was 9.2 years (±5.9), 56.4% were male, 94.8% had an updated immunization history, and 65.8% had extra vaccines. Of these children, 22.1% had comorbidities. Among the caregivers, 83.3% were mothers, the mean age was 39.4 years (±9.4), and 26.9% had attended university. The vaccine was considered safe by 61.5%, and 34.6% answered they did not know whether to consider it safe or not. Information about the vaccine was obtained through television in 84.6%, social networks in 42.3% and 34.6% in the information given by health professionals. Regarding the intention to vaccinate their children, 76.9% answered "yes", 7.7% did not answer and 15.4% answered "no". The vaccine's ineffectiveness (n=5) and inappropriate age (n=3) were the most cited reasons not to vaccinate. In 82.9%, the number of doses of the vaccine would not influence the decision. Conclusions: The study results show that caregivers have considerable resistance to the vaccination of children and adolescents against COVID-19, mainly based on the belief in the vaccine's ineffectiveness. The majority obtained information about the vaccine in the media, which reinforces the importance and the opportunity for intervention by transmitting credible and perceptible information in these media.

Clonal Hematopoiesis of Indeterminate Potential Is Associated with Increased Age-Independent Morbidity and Mortality in Patients with COVID-19- the Beyond DNA COVID-19 Project

Background: SARS-Cov-2 infections are associated with increased mortality and morbidity, largely due to inflammatory cascades and cytokine release syndrome (CRS). Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the presence and subsequent expansion of somatic, leukemia-associated driver mutations in apparently healthy individuals with normal blood counts. CHIP has been associated with increased inflammation, with cytokines such as IL1-b, IL6 and TNF-a being elevated at baseline in affected individuals. We hypothesized that the presence of CHIP in patients with COVID-19 would result in excessive inflammation-related mortality and morbidity. Methods: We used the Mayo Clinic COVID-19 database to identify patients with COVID-19 on whom peripheral blood mononuclear cells (PBMC) were available for research use (IRB approved). We carried out target-capture next generation sequencing for 220 CH related genes, by previously described methods (1000 x coverage, variant allele fraction/VAF detection limit >0.5%;Kusne Y et al AJH 2021). CHIP was defined by the presence of a CH mutation with a VAF>1% in an individual with normal baseline blood counts. Demographics, blood counts, and inflammatory markers (CRP and cytokine levels- ELISA assay) at COVID-19 diagnosis and during follow-up (as clinically indicated) were collected. COVID-19 disease severity was classified based on the presence and severity of CRS, graded using the Penn Grading Scale (Porter et. al., 2018), and the WHO ordinal scale (WHO Blueprint, 2020). We used Fisher's exact test and the Wilcoxon rank sum test to compare categorical and continuous variables. Survival analysis was performed using the Kaplan-Meier method. We accounted for differences in age and sex using multivariable-adjusted proportional hazards regression models. Results: Seventy-two CHIP mutations were detected in 56 (25%) of the 227 patients with COVID-19 that had PBMC available;median age 69 years (range;42-99 years), 61% male. Fifteen (26%) patients had 2 CHIP mutations, while 1 patient had 3 CHIP mutations. Common mutations encountered included DNMT3A (32%), TET2 (19%), SF3B1 (8%), ASXL1 (6%), MPL (5%), and TP53 (5%;Figure 1A). COVID-19 patients with CHIP were older in age (median 69 vs 57 years;p<0.0001) and had higher baseline MCP-1 (p=0.04) levels. However, there were no differences in sex, comorbidities, blood counts, IL1-b, IL6 and TNF-a levels between the two groups. The median follow-up for the entire cohort was 9 months. The relative change from baseline in blood counts and inflammatory markers (CRP and cytokines) during follow-up was similar in CHIP and non-CHIP patients, with the exception that COVID-19-onset neutropenia was more common in CHIP patients (8% vs 1%;p=0.017) compared to those without CHIP. At last follow up neutropenia had resolved in all patients. Both groups had comparable number of patients with CRS (61% CHIP vs 53% non-CHIP patients, p=0.354, Figure 1B), however, CHIP patients had more severe CRS (median Penn Grade 3 versus 2 in non-CHIP, p=0.018, Figure 1C). Based on the WHO ordinal scale, CHIP patients were more likely to experience hospitalization with severe disease and death (61% versus 45% in non-CHIP, p = 0.049). Moreover, COVID-19 CHIP patients experienced worse overall survival in comparison to patients without CHIP (median 13.1 months vs not reached, p<0.001, Figure 1D). This association remained consistent after adjusting for age and sex at the time of COVID-19 diagnosis (HR 3.15, 95% 1.32 - 7.55, p = 0.010). At last follow-up, 22 deaths were documented: 13 (23%) in patients with CHIP and 9 (5%) in the non-CHIP group (p=0.02), with the primary cause for mortality being hypoxic respiratory failure (62% in CHIP vs 44% non-CHIP, p=0.04). Conclusions: In this study, we observe an age-independent impact of CHIP on COVID-19 associated inflammatory morbidity (CRS) and mortality (hypoxemic respiratory failure). We are currently carrying out detailed single cell (ssDNA, RNA and ATAC-seq) and proteomic studies (O-link PEA assays) to better elucidate this pathophysiology. [Formula presented] Disclosures: Patnaik: Kura Oncology: Research Funding;StemLine: Research Funding.

Archiving the pandemic - historian's projects and "duty to memory"

The years 2020 and 2021 brought significant changes to our lives. New habits were introduced in everyday life from the need for social isolation and the way we face death was also radically affected. The article seeks to reflect on the fundamental role of the historian in recording the pandemic, based on the presentation of four works produced in this period that directly related the archive, memory, and the Covid-19 pandemic. The debate revolves around the possibility of reflecting on the "duty of memory" and the production of documents that may, in the future, help us understand how Brazil reached the level of more than half a million deaths from the disease in so little. time.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

The First 100 Days of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Control in Vietnam.

BACKGROUND: One hundred days after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Vietnam on 23 January, 270 cases were confirmed, with no deaths. We describe the control measures used by the government and their relationship with imported and domestically acquired case numbers, with the aim of identifying the measures associated with successful SARS-CoV-2 control. METHODS: Clinical and demographic data on the first 270 SARS-CoV-2 infected cases and the timing and nature of government control measures, including numbers of tests and quarantined individuals, were analyzed. Apple and Google mobility data provided proxies for population movement. Serial intervals were calculated from 33 infector-infectee pairs and used to estimate the proportion of presymptomatic transmission events and time-varying reproduction numbers. RESULTS: A national lockdown was implemented between 1 and 22 April. Around 200 000 people were quarantined and 266 122 reverse transcription polymerase chain reaction (RT-PCR) tests conducted. Population mobility decreased progressively before lockdown. In total, 60% (163/270) of cases were imported; 43% (89/208) of resolved infections remained asymptomatic for the duration of infection. The serial interval was 3.24 days, and 27.5% (95% confidence interval [CI], 15.7%-40.0%) of transmissions occurred presymptomatically. Limited transmission amounted to a maximum reproduction number of 1.15 (95% CI, .·37-2.·36). No community transmission has been detected since 15 April. CONCLUSIONS: Vietnam has controlled SARS-CoV-2 spread through the early introduction of mass communication, meticulous contact tracing with strict quarantine, and international travel restrictions. The value of these interventions is supported by the high proportion of asymptomatic and imported cases, and evidence for substantial presymptomatic transmission.

Protocol for the Implementation and Assessment of "MoodUP": A Stepped Care Model Assisted by a Digital Platform to Accelerate Access to Mental Health Care for Cancer Patients Amid the COVID-19 Pandemic

The COVID-19 pandemic has important consequences for the mental health of populations. Patients with cancer, already at risk for poor mental health outcomes, are not expected to be spared from these consequences, prompting the need for health services to improve responsiveness. This article presents the research protocol for an implementation study designed to describe the uptake of a well-studied and recognized system for the treatment of depression and anxiety (Stepped-care) during the specific context of a Pandemic in an oncological site. The system set-up will be assisted by a digital platform (MoodUP), where patients undergoing cancer treatment will be screened for anxiety and depressive symptoms, triaged by severity level and algorithm-matched to recommended interventions. Patients undergoing cancer treatment at a cancer clinic in Portugal will be invited to subscribe to the MoodUP platform where they will complete a self-reported questionnaire (Hospital Anxiety and Depression Scale) to screen their anxiety and depressive symptoms. Data will be instantly collected, and an algorithm will activate severity-matched intervention suggestions, through a case manager that will coordinate care. The specific objectives of this study will be to describe the implementation and acceptability of the care system by patients and staff, the barriers to and facilitators of implementation, the proportion of patients accessing the system and their pathways through the various stepped-care interventions, and patient perceptions regarding the feasibility and appropriateness of the eHealth platform. Moreover, exploratory analyses will be conducted to describe patterns of anxiety and depression symptoms variation across all patients, as well as within sociodemographically, clinically and contextually characterized subgroups, to characterize their care needs and access, as well as to explore for whom the MoodUP care system may be more appropriate. This study is expected to improve processes for collaborative mental healthcare in oncology and accelerate the digitalization of services, towards the improvement of mental healthcare access, and management of high-risk patients, during the COVID-19 pandemic.

LY-CoV1404 potently neutralizes SARS-CoV-2 variants (preprint)

LY-CoV1404 is a highly potent, neutralizing, SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody identified from a convalescent COVID-19 patient approximately 60 days after symptom onset. In pseudovirus studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.427/B.1.429, P.1, and B.1.526 and binds to these variants in the presence of their underlying RBD mutations (which include K417N, L452R, E484K, and N501Y). LY-CoV1404 also neutralizes authentic SARS-CoV-2 in two different assays against multiple isolates. The RBD positions comprising the LY-CoV1404 epitope are highly conserved, with the exception of N439 and N501; notably the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of variant binding, potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 is one in a panel of well-characterized, clinically developable antibodies that could be deployed rapidly to address current and emerging variants. New variant-resistant treatments such as LY-CoV1404 are desperately needed, given that some of the existing therapeutic antibodies are less effective or ineffective against certain variants and the impact of variants on vaccine efficacy is still poorly understood.

Novel Mutation of SARS-CoV-2, Vietnam, July 2020.

A cluster of severe acute respiratory syndrome coronavirus 2 infections in Danang, Vietnam, began July 25, 2020, and resulted in 551 confirmed cases and 35 deaths as of February 2021. We analyzed 26 sequences from this cluster and identified a novel shared mutation in nonstructural protein 9, suggesting a single introduction into Vietnam.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. ONE SENTENCE SUMMARY: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

SARS-CoV-2 Spike protein promotes hyper-inflammatory response that can be ameliorated by Spike-antagonistic peptide and FDA-approved ER stress and MAP kinase inhibitors in vitro (preprint)

SARS-CoV-2 infection causes an inflammatory cytokine storm and acute lung injury. Currently there are no effective antiviral and/or anti-inflammatory therapies. Here we demonstrate that 2019 SARS-CoV-2 spike protein subunit 1 (CoV2-S1) induces high levels of NF-{kappa}B activations, production of pro-inflammatory cytokines and mild epithelial damage, in human bronchial epithelial cells. CoV2-S1-induced NF-{kappa}B activation requires S1 interaction with human ACE2 receptor and early activation of endoplasmic reticulum (ER) stress, and associated unfolded protein response (UPR), and MAP kinase signalling pathways. We developed an antagonistic peptide that inhibits S1-ACE2 interaction and CoV2-S1-induced productions of pro-inflammatory cytokines. The existing FDA-approved ER stress inhibitor, 4-phenylburic acid (4-PBA), and MAP kinase inhibitors, trametinib and ulixertinib, ameliorated CoV2-S1-induced inflammation and epithelial damage. These novel data highlight the potentials of peptide-based antivirals for novel ACE2-utilising CoVs, while repurposing existing drugs may be used as treatments to dampen elevated inflammation and lung injury mediated by SARS-CoV-2.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection (preprint)

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection.

SARS-CoV-2 viral budding and entry can be modeled using virus-like particles (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first discovered in December 2019 in Wuhan, China and expeditiously spread across the globe causing a global pandemic. While a select agent designation has not been made for SARS-CoV-2, closely related SARS-CoV-1 and MERS coronaviruses are classified as Risk Group 3 select agents, which restricts use of the live viruses to BSL-3 facilities. Such BSL-3 classification make SARS-CoV-2 research inaccessible to the majority of functioning research laboratories in the US; this becomes problematic when the collective scientific effort needs to be focused on such in the face of a pandemic. In this work, we assessed the four structural proteins from SARS-CoV-2 for their ability to form virus-like particles (VLPs) from human cells to form a competent system for BSL-2 studies of SARS-CoV-2. Herein, we provide methods and resources of producing, purifying, fluorescently and APEX2-labeling of SARS-CoV-2 VLPs for the evaluation of mechanisms of viral budding and entry as well as assessment of drug inhibitors under BSL-2 conditions.

Thiopurines activate an antiviral unfolded protein response that blocks viral glycoprotein accumulation in cell culture infection model (preprint)

Enveloped viruses utilize the host cell secretory pathway to synthesize viral glycoproteins and direct them to sites of assembly. Using an image-based screen, we identified two thiopurines, 6-thioguanine (6-TG) and 6-thioguanosine (6-TGo), that selectively disrupted the processing and accumulation of influenza A virus glycoproteins hemagglutinin (HA) and neuraminidase (NA). Selective disruption of IAV glycoprotein processing and accumulation by 6-TG and 6-TGo correlated with unfolded protein response (UPR) activation. 6-TG and 6-TGo also inhibited replication of the human coronavirus OC43 (HCoV-OC43), which correlated with UPR/ISR activation and diminished accumulation of ORF1ab and nucleocapsid (N) mRNAs, which suggests broader disruption of coronavirus gene expression in ER-derived cytoplasmic compartments. The chemically similar thiopurine 6-mercaptopurine (6-MP) had little effect on the UPR and did not affect IAV or HCoV-OC43 replication. Consistent with reports on other CoV Spike (S) proteins, ectopic expression of SARS-CoV-2 S protein caused UPR activation. 6-TG inhibited accumulation of full length S0 or furin-cleaved S2 fusion proteins, but spared the S1 ectodomain. DBeQ, which inhibits the p97 AAA-ATPase required for retrotranslocation of ubiquitinated misfolded proteins during ER-associated degradation (ERAD) restored accumulation of S0 and S2 proteins in the presence of 6-TG, suggesting that 6-TG induced UPR accelerates ERAD-mediated turnover of membrane-anchored S0 and S2 glycoproteins. Taken together, these data indicate that 6-TG and 6-TGo are effective host-targeted antivirals that trigger the UPR and disrupt accumulation of viral glycoproteins. Importantly, our data demonstrate for the first time the efficacy of these thiopurines in limiting IAV and HCoV-OC43 replication in cell culture models.