Prevalence of Depression, Anxiety, Post-Traumatic Stress, and Insomnia Symptoms Among Frontline Healthcare Workers in a COVID-19 Hospital in Northeast Mexico.

OBJECTIVE: Frontline healthcare workers (FHCWs) exposed to COVID-19 patients are at an increased risk of developing psychological burden. This study aims to determine the prevalence of mental health symptoms and associated factors among Mexican FHCWs attending COVID-19 patients. METHODS: FHCWs, including attending physicians, residents/fellows, and nurses providing care to COVID-19 patients at a private hospital in Monterrey, Mexico, were invited to answer an online survey between August 28, and November 30, 2020. Symptoms of depression, anxiety, post-traumatic stress, and insomnia were evaluated with the Patient Health Questionnaire (PHQ)-9, Generalized Anxiety Disorder (GAD)-7, Impact of Event Scale-Revised (IES-R), and Insomnia Severity Index (ISI). Multivariate analysis was performed to identify variables associated with each outcome. RESULTS: 131 FHCWs, 43.5% attending physicians, 19.8% residents/fellows, and 36.6% nurses were included. The overall prevalence of depression, anxiety, post-traumatic stress, and insomnia was 36%, 21%, 23%, and 24% respectively. Multivariate analysis revealed that residents/fellows and nurses reported more depression and insomnia than attending physicians. Although not significant, residents/fellows were more likely to experience all symptoms than nurses. CONCLUSIONS: Mexican FHCWs, especially nurses and residents/fellows, experienced a significant psychological burden while attending to COVID-19 patients. Tailored interventions providing support to FHCWs during future outbreaks are required.

Efficient capture of recombinant SARS-CoV-2 receptor-binding domain (RBD) with citrate-coated magnetic iron oxide nanoparticles.

Several vaccines against COVID-19 use a recombinant SARS-CoV-2 receptor-binding domain (RBD) as antigen, making the purification of this protein a key step in their production. In this work, citrate-coated magnetic iron oxide nanoparticles were evaluated as nano adsorbents in the first step (capture) of the purification of recombinant RBD. The nanoparticles were isolated through coprecipitation and subsequently coated with sodium citrate. The citrate-coated nanoparticles exhibited a diameter of 10 ± 2 nm, a hydrodynamic diameter of 160 ± 3 nm, and contained 1.9 wt% of citrate. The presence of citrate on the nanoparticles' surface was confirmed through FT-IR spectra and thermogravimetric analysis. The crystallite size (10.1 nm) and the lattice parameter (8.3646 Å) were determined by X-ray diffraction. In parallel, RBD-containing supernatant extracted from cell culture was exchanged through ultrafiltration and diafiltration into the adsorption buffer. The magnetic capture was then optimized using different concentrations of nanoparticles in the purified supernatant, and we found 40 mg mL-1 to be optimal. The ideal amount of nanoparticles was assessed by varying the RBD concentration in the supernatant (between 0.113 mg mL-1 and 0.98 mg mL-1), which resulted in good capture yields (between 83 ± 5% and 94 ± 4%). The improvement of RBD purity after desorption was demonstrated by SDS-PAGE and RP-HPLC. Furthermore, the magnetic capture was scaled up 100 times, and the desorption was subjected to chromatographic purifications. The obtained products recognized anti-RBD antibodies and bound the ACE2 receptor, proving their functionality after the developed procedure.

Human ACE2 expression, a major tropism determinant for SARS-CoV-2, is regulated by upstream and intragenic elements.

Angiotensin-converting enzyme 2 (ACE2), part of the renin-angiotensin system (RAS), serves as an entry point for SARS-CoV-2, leading to viral proliferation in permissive cell types. Using mouse lines in which the Ace2 locus has been humanized by syntenic replacement, we show that regulation of basal and interferon induced ACE2 expression, relative expression levels of different ACE2 transcripts, and sexual dimorphism in ACE2 expression are unique to each species, differ between tissues, and are determined by both intragenic and upstream promoter elements. Our results indicate that the higher levels of expression of ACE2 observed in the lungs of mice relative to humans may reflect the fact that the mouse promoter drives expression of ACE2 in populous airway club cells while the human promoter drives expression in alveolar type 2 (AT2) cells. In contrast to transgenic mice in which human ACE2 is expressed in ciliated cells under the control of the human FOXJ1 promoter, mice expressing ACE2 in club cells under the control of the endogenous Ace2 promoter show a robust immune response after infection with SARS-CoV-2, leading to rapid clearance of the virus. This supports a model in which differential expression of ACE2 determines which cell types in the lung are infected, and this in turn modulates the host response and outcome of COVID-19.

Broadly neutralizing anti-S2 antibodies protect against all three human betacoronaviruses that cause deadly disease.

Pan-betacoronavirus neutralizing antibodies may hold the key to developing broadly protective vaccines against novel pandemic coronaviruses and to more effectively respond to SARS-CoV-2 variants. The emergence of Omicron and subvariants of SARS-CoV-2 illustrates the limitations of solely targeting the receptor-binding domain (RBD) of the spike (S) protein. Here, we isolated a large panel of broadly neutralizing antibodies (bnAbs) from SARS-CoV-2 recovered-vaccinated donors, which targets a conserved S2 region in the betacoronavirus spike fusion machinery. Select bnAbs showed broad in vivo protection against all three deadly betacoronaviruses, SARS-CoV-1, SARS-CoV-2, and MERS-CoV, which have spilled over into humans in the past two decades. Structural studies of these bnAbs delineated the molecular basis for their broad reactivity and revealed common antibody features targetable by broad vaccination strategies. These bnAbs provide new insights and opportunities for antibody-based interventions and for developing pan-betacoronavirus vaccines.

Erythema nodosum after COVID-19 vaccine: Report of two pediatric cases.

With the introduction of large-scale COVID-19 vaccination programs, a variety of cutaneous manifestations have been described. We present two girls (ages 12 and 5 years) who developed erythema nodosum (EN) 3 and 14 days after Pfizer-BioNTech COVID-19 vaccination, respectively. While EN after COVID-19 vaccination has been reported in adults, it is can also occur in children.

Immunogenicity and protective efficacy of a rhesus adenoviral vaccine targeting conserved COVID-19 replication transcription complex.

The COVID-19 pandemic marks the third coronavirus pandemic this century (SARS-CoV-1, MERS, SARS-CoV-2), emphasizing the need to identify and evaluate conserved immunogens for a pan-sarbecovirus vaccine. Here we investigate the potential utility of a T-cell vaccine strategy targeting conserved regions of the sarbecovirus proteome. We identified the most conserved regions of the sarbecovirus proteome as portions of the RNA-dependent RNA polymerase (RdRp) and Helicase proteins, both of which are part of the coronavirus replication transcription complex (RTC). Fitness constraints suggest that as SARS-CoV-2 continues to evolve these regions may better preserve cross-reactive potential of T-cell responses than Spike, Nucleocapsid, or Membrane proteins. We sought to determine if vaccine-elicited T-cell responses to the highly conserved regions of the RTC would reduce viral loads following challenge with SARS-CoV-2 in mice using a rhesus adenovirus serotype 52 (RhAd52) vector. The RhAd52.CoV.Consv vaccine generated robust cellular immunity in mice and led to significant reductions in viral loads in the nasal turbinates following challenge with a mouse-adapted SARS-CoV-2. These data suggest the potential utility of T-cell targeting of conserved regions for a pan-sarbecovirus vaccine.

Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine.

Coronavirus vaccines that are highly effective against current and anticipated SARS-CoV-2 variants are needed to control COVID-19. We previously reported a receptor-binding domain (RBD)-sortase A-conjugated ferritin nanoparticle (scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected non-human primates (NHPs) from SARS-CoV-2 WA-1 infection. Here, we find the RBD-scNP induced neutralizing antibodies in NHPs against pseudoviruses of SARS-CoV and SARS-CoV-2 variants including 614G, Beta, Delta, Omicron BA.1, BA.2, BA.2.12.1, and BA.4/BA.5, and a designed variant with escape mutations, PMS20. Adjuvant studies demonstrate variant neutralization titers are highest with 3M-052-aqueous formulation (AF). Immunization twice with RBD-scNPs protect NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protect mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect animals from multiple different SARS-related viruses. Such a vaccine could provide broad immunity to SARS-CoV-2 variants.

A Multitrait Locus Regulates Sarbecovirus Pathogenesis.

Infectious diseases have shaped the human population genetic structure, and genetic variation influences the susceptibility to many viral diseases. However, a variety of challenges have made the implementation of traditional human Genome-wide Association Studies (GWAS) approaches to study these infectious outcomes challenging. In contrast, mouse models of infectious diseases provide an experimental control and precision, which facilitates analyses and mechanistic studies of the role of genetic variation on infection. Here we use a genetic mapping cross between two distinct Collaborative Cross mouse strains with respect to severe acute respiratory syndrome coronavirus (SARS-CoV) disease outcomes. We find several loci control differential disease outcome for a variety of traits in the context of SARS-CoV infection. Importantly, we identify a locus on mouse chromosome 9 that shows conserved synteny with a human GWAS locus for SARS-CoV-2 severe disease. We follow-up and confirm a role for this locus, and identify two candidate genes, CCR9 and CXCR6, that both play a key role in regulating the severity of SARS-CoV, SARS-CoV-2, and a distantly related bat sarbecovirus disease outcomes. As such we provide a template for using experimental mouse crosses to identify and characterize multitrait loci that regulate pathogenic infectious outcomes across species. IMPORTANCE Host genetic variation is an important determinant that predicts disease outcomes following infection. In the setting of highly pathogenic coronavirus infections genetic determinants underlying host susceptibility and mortality remain unclear. To elucidate the role of host genetic variation on sarbecovirus pathogenesis and disease outcomes, we utilized the Collaborative Cross (CC) mouse genetic reference population as a model to identify susceptibility alleles to SARS-CoV and SARS-CoV-2 infections. Our findings reveal that a multitrait loci found in chromosome 9 is an important regulator of sarbecovirus pathogenesis in mice. Within this locus, we identified and validated CCR9 and CXCR6 as important regulators of host disease outcomes. Specifically, both CCR9 and CXCR6 are protective against severe SARS-CoV, SARS-CoV-2, and SARS-related HKU3 virus disease in mice. This chromosome 9 multitrait locus may be important to help identify genes that regulate coronavirus disease outcomes in humans.

Potent and broad neutralization of SARS-CoV-2 variants of concern (VOCs) including omicron sub-lineages BA.1 and BA.2 by biparatopic human VH domains.

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics with high neutralization breadth. Here, we characterized a human VH domain, F6, which we generated by sequentially panning large phage-displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that broadly and potently neutralized VOCs including Omicron. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 variants including Omicron and highlight a vulnerable epitope within the spike that may be exploited to achieve broad protection against circulating variants.

Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses.

The emergence of current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed. Here, we utilized a targeted donor selection strategy to isolate a large panel of human broadly neutralizing antibodies (bnAbs) to sarbecoviruses. Many of these bnAbs are remarkably effective in neutralizing a diversity of sarbecoviruses and against most SARS-CoV-2 VOCs, including the Omicron variant. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor-binding domain (RBD). Consistent with targeting of conserved sites, select RBD bnAbs exhibited protective efficacy against diverse SARS-like coronaviruses in a prophylaxis challenge model in vivo. These bnAbs provide new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and provide a molecular basis for effective design of pan-sarbecovirus vaccines.

Transient breast livedo reticularis-like exanthem in an adolescent with COVID-19.

Livedo reticularis-like eruptions have been described in different viral infections. In patients with COVID-19, livedo reticularis-like rashes are usually mild, typically present in a symmetric distribution and mostly involve the lower limbs. A case of livedo reticularis located exclusively on the breasts of a girl with mild systemic symptoms of COVID-19 is presented. Coagulation studies were normal and findings disappeared within 1 week.

Longitudinal Remote SBRT/SRS Training in Latin America: A Prospective Cohort Study

Background Continuing medical education in stereotactic technology are scarcely accessible in developing countries. We report the results of upscaling a longitudinal telehealth training course on stereotactic body radiation therapy (SBRT) and stereotactic radiosurgery (SRS), after successfully developing a pilot course in Latin America. Methods Longitudinal training on SBRT and SRS was provided to radiation oncology practitioners in Peru and Colombia at no cost. The program included sixteen weekly 1-hour live conferencing sessions with interactive didactics and a cloud-based platform for case-based learning. Participant-reported confidence was measured in 16 SBRT/SRS practical domains, based on a 1-to-5 Likert scale. Pre- and post-curriculum exams were required for participation credit. Knowledge-baseline, pre- and post-curriculum surveys, overall and single professional-group confidence changes, and exam results were assessed. Results One hundred and seventy-three radiotherapy professionals participated. An average of 56 (SD ±18) attendees per session were registered. Fifty (29.7%) participants completed the pre- and post-curriculum surveys, of which 30% were radiation oncologists (RO), 26% radiation therapists (RTT), 20% residents, 18% medical physicists and 6% neurosurgeons. Significant improvements were found across all 16 domains with overall mean +0.55 (SD ±0.17, p<0.001) Likert-scale points. Significant improvements in individual competences were most common among medical physicists, RTT and residents. Pre- and post-curriculum exams yielded a mean 16.15/30 (53.8 ± 20.3%) and 23.6/30 (78.7 ± 19.3%) correct answers (p<0.001). Conclusion Longitudinal telehealth training is an effective method for improving confidence and knowledge on SBRT/SRS amongst professionals. Remote continuing medical education should be widely adopted in lower-middle income countries.

Therapeutic treatment with an oral prodrug of the remdesivir parental nucleoside is protective against SARS-CoV-2 pathogenesis in mice.

The coronavirus disease 2019 (COVID-19) pandemic remains uncontrolled despite the rapid rollout of safe and effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, underscoring the need to develop highly effective antivirals. In the setting of waning immunity from infection and vaccination, breakthrough infections are becoming increasingly common and treatment options remain limited. In addition, the emergence of SARS-CoV-2 variants of concern, with their potential to escape neutralization by therapeutic monoclonal antibodies, emphasizes the need to develop second-generation oral antivirals targeting highly conserved viral proteins that can be rapidly deployed to outpatients. Here, we demonstrate the in vitro antiviral activity and in vivo therapeutic efficacy of GS-621763, an orally bioavailable prodrug of GS-441524, the parent nucleoside of remdesivir, which targets the highly conserved virus RNA-dependent RNA polymerase. GS-621763 exhibited antiviral activity against SARS-CoV-2 in lung cell lines and two different human primary lung cell culture systems. GS-621763 was also potently antiviral against a genetically unrelated emerging coronavirus, Middle East respiratory syndrome CoV (MERS-CoV). The dose-proportional pharmacokinetic profile observed after oral administration of GS-621763 translated to dose-dependent antiviral activity in mice infected with SARS-CoV-2. Therapeutic GS-621763 administration reduced viral load and lung pathology; treatment also improved pulmonary function in COVID-19 mouse model. A direct comparison of GS-621763 with molnupiravir, an oral nucleoside analog antiviral that has recently received EUA approval, proved both drugs to be similarly efficacious in mice. These data support the exploration of GS-441524 oral prodrugs for the treatment of COVID-19.

Evolution of incidence of chilblain-like lesions in children during the first year of COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic has brought innumerable reports of chilblains. The relation between pernio-like acral eruptions and COVID-19 has not been fully elucidated because most reported cases have occurred in patients with negative microbiological tests for SARS-CoV-2. METHODS: A retrospective study of 49 cases of chilblains seen during the first year of the pandemic in a children's hospital in Madrid, Spain. The incidence of these skin lesions was correlated with the number of COVID-19 admissions and environmental temperatures. Patients were separated into two groups depending on the day of onset (strict lockdown period vs. outside the lockdown period). RESULTS: Most chilblains cases presented during the first and third waves of the pandemic, paralleling the number of COVID-19 admissions. The first wave coincided with a strict lockdown, and the third wave coincided with the lowest ambient seasonal temperatures of the year. Systemic symptoms preceding chilblains were more frequent in the first wave (45.8% vs. 8.0%, p = .002), as was the co-occurrence with erythema multiforme-like lesions (16.7% vs. 0%, p = .033). Laboratory test and skin biopsies were performed more frequently in the first wave (75.0% vs. 12.0%, p < .001; and 25.0% vs. 0%, p = .007; respectively). Five patients developed recurrent cutaneous symptoms. CONCLUSIONS: An increased incidence of chilblains coincided not only with the two major waves of the pandemic, but also with the strict lockdown period in the first wave and low seasonal temperatures during the third wave. Both increased sedentary behaviors and cold environmental temperatures may have played an additive role in the development of COVID-19-related chilblains.

Modeling and control of an invasive mechanical ventilation system using the active disturbances rejection control structure.

We propose a mandatory invasive mechanical ventilator prototype for severe COVID-19 patients with volume and pressure control operation modes. This system comprises basic pneumatic elements and sensors. Its performance is similar to commercial equipment, and it presents robustness to external disturbances and parametric uncertainties. To develop a control strategy, we propose a mathematical model with a variable structure that incorporates the dead zone phenomenon of the proportional valve, and considers external disturbances and parametric uncertainties. Based on this model, we propose a global control strategy that is based on pressure and flow regulation controllers, which use the active disturbances rejection control structure (ADRC). In this strategy, we propose robust state observers to estimate disturbances and the signals necessary for implementing the controllers. We illustrate the performance of the prototype and the control strategy through numerical simulations and experiments. We also compare its performance with PID controllers. These results corroborate its effectiveness and the possibility of its application in invasive mechanical ventilators with a simple structure, which can significantly help critical care of COVID-19 inpatients.

Ultrapotent antibodies against diverse and highly transmissible SARS-CoV-2 variants.

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOCs) that are resistant to therapeutic antibodies highlights the need for continuing discovery of broadly reactive antibodies. We identified four receptor binding domain-targeting antibodies from three early-outbreak convalescent donors with potent neutralizing activity against 23 variants, including the B.1.1.7, B.1.351, P.1, B.1.429, B.1.526, and B.1.617 VOCs. Two antibodies are ultrapotent, with subnanomolar neutralization titers [half-maximal inhibitory concentration (IC50) 0.3 to 11.1 nanograms per milliliter; IC80 1.5 to 34.5 nanograms per milliliter). We define the structural and functional determinants of binding for all four VOC-targeting antibodies and show that combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting their potential in mitigating resistance development.

Optimization of non-coding regions for a non-modified mRNA COVID-19 vaccine.

The CVnCoV (CureVac) mRNA vaccine for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was recently evaluated in a phase 2b/3 efficacy trial in humans1. CV2CoV is a second-generation mRNA vaccine containing non-modified nucleosides but with optimized non-coding regions and enhanced antigen expression. Here we report the results of a head-to-head comparison of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in non-human primates. We immunized 18 cynomolgus macaques with two doses of 12 µg lipid nanoparticle-formulated CVnCoV or CV2CoV or with sham (n = 6 per group). Compared with CVnCoV, CV2CoV induced substantially higher titres of binding and neutralizing antibodies, memory B cell responses and T cell responses as well as more potent neutralizing antibody responses against SARS-CoV-2 variants, including the Delta variant. Moreover, CV2CoV was found to be comparably immunogenic to the BNT162b2 (Pfizer) vaccine in macaques. Although CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded more robust protection with markedly lower viral loads in the upper and lower respiratory tracts. Binding and neutralizing antibody titres were correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of a non-modified mRNA SARS-CoV-2 vaccine in non-human primates.