ABSTRACT
Background: The continued emergence of severe acute respiratory syndrome coronaviruses (SARS-CoVs) and recent explosion of the SARS-CoV-2 pandemic highlights the need for broad and potent antibody recognition and understanding the contexts in which they may develop. Antibodies with cross reactivity across SARS lineages may be of particular value in preparing for future outbreaks of new sarbecoviruses. Method(s): We isolated monoclonal antibodies (mAbs) from an individual 60-days post-vaccination, 30-days post Delta-infection. Reconstructed antibodies were screened for binding to a panel of prefusion-stabilized Spike trimers from SARS-CoV-2 and other beta-coronaviruses using enzyme-linked immunosorbent assay (ELISA). Neutralization potency and breadth was assessed using a spike-pseudotyped lentivirus neutralization assay. Additionally, epitope and escape mutant profiling was conducted by deep mutational scanning (DMS) to identify mutations that affect antibody binding. Lastly, binding breadth was further evaluated using a yeast display library of RBDs from SARS-CoV-2 variants and related sarbecoviruses. Result(s): We identified several SARS-CoV-2-specific mAbs that neutralized SARS-CoV-2 variants of concern (VOCs) and SARS-CoV-1. Notably, two of these mAbs (C68.61 and C68.185) neutralized SARS-CoV-1 with an IC50 = 307 and 139 ng/mL (respectively) that is similar to or better than the potency of S309 (IC50 = 206 ng/mL) and CR3022 (IC50 = 981 ng/mL), which are mAbs isolated from individuals with SARS-CoV-1 infections. C68.61 also neutralized all Omicron VOCs tested and retained neutralization activity against currently circulating variants BQ1.1 (IC50=790 ng/ml) and XBB (IC50=590 ng/ml). Key C68.61 mAbescape mutations identified by DMS in the Omicron BA.2 background yeast display library included sites K462, E465, R466, and I468, which are conserved sites across all VOCs and SARS-CoV-1. The isolated mAbs displayed crossreactive binding to RBDs from diverse SARS-CoV-1-related CoVs and African and European sarbecovirus isolates as well as SARS-CoV-2 VOCs. Conclusion(s): Here we describe mAbs from a SARS-CoV-2-infected individual that bound and neutralized both SARS-CoV-2 and SARS-CoV-1, including one that showed breadth across recent VOCs. Given their breadth, these SARS-CoV-2 cross-reactive mAbs may be robust to viral escape and thus could contribute to therapeutic efforts. In addition, these mAbs displayed broad cross-reactive activity across sarbecoviruses and may be beneficial against future spillover events.
ABSTRACT
Background: While remarkable and rapid progress was made in fighting the SARS-CoV-2 pandemic with vaccines and therapeutic antibodies, these approaches were quickly compromised by viral evolution. Therapeutic monoclonal antibodies (mAbs) that were once authorized for clinical use, which all target the receptor binding domain (RBD), are no longer effective against current variants of concern (VOCs) due to mutations in this region of Spike. Thus, to achieve durable protection against SARS-CoV-2, novel mAbs need to show breadth and potency across VOCs and target epitopes that are more constrained. Method(s): mAbs from an individual who had a breakthrough Delta VOC infection after vaccination were isolated from Spike-specific memory B cells. mAbs were assessed for binding affinity and neutralization potency using Spike-pseudotyped lentivirus (PSV) and live SARS-CoV-2 virus neutralization assays. Epitopes were mapped using deep mutational sequencing (DMS) and structural-based methods. Result(s): Three novel mAbs (C68.3, C68.13, C68.59) demonstrated binding breadth to Spikes from various VOCs including Omicron VOCs despite that C68 had not yet been exposed to Omicron. These mAbs potently neutralized the Wuhan-Hu-1 vaccine and Delta strains (IC50 = 9-61ng/mL), and early Omicron strains BA.1, BA.2, BA.5 (IC50 = 12-149 ng/mL). C68.3 and C68.59 retained potency against recent VOCs BQ.1.1 and XBB (IC50 = 121-122 ng/mL and 56-82 ng/mL, respectively) in the PSV assay. Similar neutralization activity was observed in the live virus assay. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs previously authorized for treatment and they showed greater breadth. The mAbs target distinct epitopes on the Spike glycoprotein, two in the RBD (C68.3, C68.13) and one in an invariant region downstream of RBD in subdomain 1 (SD1) (C68.59). Structural analysis of C68.59 Fab binding to Spike trimer revealed significant allosteric changes to regions of Spike outside of the epitope in the S2 unit. Finally, DMS escape pathways showed these mAbs target regions highly conserved across VOCs that are also functionally constrained, suggesting escape could incur a fitness cost. Conclusion(s): Overall, these mAbs are novel in their breadth across VOCs and include a potent mAb targeting a rare epitope outside of the RBD in SD1. These mAbs focus on diverse, functionally constrained regions in Spike making them candidates for development as combination therapeutics with good durability against future VOCs.
ABSTRACT
Background: The Extracorporeal Treatments in Poisoning (EXTRIP) workgroup provides a weak conditional recommendation in support of hemodialysis (HD) for select patients with severe phenytoin poisoning. Despite this recommendation, the HD clearance of phenytoin is poorly studied. We present a patient who developed phenytoin toxicity that was treated with hemodialysis and report on the efficacy of phenytoin removal during HD. Case report: An 87-year-old man with epilepsy who was maintained on a stable dose of 300mg phenytoin extended-release daily was admitted to the hospital for treatment of Coronavirus Disease 2019 and congestive heart failure. On hospital day 14, the patient had a gradual onset of depressed mental status with hypothermia (nadir 35 degrees Celsius). At this time, he had a rising total blood phenytoin concentration (peak 49.3 mcg/mL [therapeutic 10-20mcg/mL] with an albumin of 3.8 g/dL [normal 3.4-5.4 g/dL]). The patient's other medications included furosemide, aspirin, atorvastatin, digoxin, doxycycline, metoprolol tartrate, and warfarin;he was also receiving albumin and crystalloid for hypovolemia (albumin nadir on hospital day 14: 2.5 g/dL). Free phenytoin concentrations were not available. Alternate etiologies of hypothermia (endocrine, infectious) were excluded. The Poison Control Center was consulted and recommended HD because of the concern for prolonged coma, as per EXTRIP guidelines. The patient received three sessions of HD over a period of 6 days at 2.5-3 h per session using an F160 Optiflux membrane filter (Fresenius Medical Care, Waltham, MA, USA), with a blood flow rate of 350mL/min and a dialysate flow rate of 700mL/min. After the first session of HD (2.5 h) on hospital day 21, his hypothermia resolved and his phenytoin concentration fell from 39.2mcg/mL to 34.2 mcg/mL with only mild improvement in his mental status. After 6 days (hospital day 27), his phenytoin concentration decreased to 19.5 mcg/mL and his mental status normalized. Effluent from the first HD session had phenytoin concentrations below the limit of detection (0.50mcg/mL). Thus, no greater than 52mg of phenytoin was removed during a 2.5-h session of hemodialysis. Discussion(s): The reason for the sudden increase in blood phenytoin concentrations in this patient is unclear in the absence of drug-drug interactions or dosing changes to the phenytoin. Although uncommonly reported, patients with phenytoin toxicity can experience hypothermia. In this case, the patient's hypothermia resolved during HD, although it is unclear if this was related to changes in phenytoin concentration or (more likely) direct extracorporeal warming via the HD machine. If the patient's phenytoin clearance from the first session were extrapolated to subsequent sessions an estimated maximum of 166.4mg of phenytoin would be removed in 8 total hours of HD, which is far less than previously reported phenytoin clearances on the order of grams. This difference may be related to the use of high cutoff dialysis membranes in prior studies, which are not routinely used. Conclusion(s): Although HD rapidly resolved this patient's hypothermia, a minimal amount of phenytoin was recovered in the patient's dialysate. Prior studies suggesting consequential clearance and efficacy of phenytoin removal by extracorporeal treatment may not apply to routine HD methods. Further studies on the utility of extracorporeal treatment for phenytoin toxicity are needed.
ABSTRACT
Advances in genome sequence have enabled rapid identification of new SARS-CoV-2 variant lineages, but a major challenge is understanding how mutations in these variants will affect anti-viral immunity. Here I describe new high-throughout assays that provide data that can be used to rapidly interpret the consequences of SARS-CoV-2 mutations.
ABSTRACT
Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection elicits antibodies (Abs) that bind several viral proteins such as the spike entry protein and the abundant nucleocapsid (N) protein. We examined convalescent sera collected through 6 months (~24wks) post-SARS-CoV-2 infection in children to evaluate changes in neutralization potency and N-binding. Methods. Outpatient, hospitalized, and community recruited volunteers < 18 years with COVID-19 were enrolled in a longitudinal study at Seattle Children's Hospital. Analysis includes symptomatic and asymptomatic children with laboratory-confirmed SARS-CoV-2 infection who provided blood samples at approximately 4wks (range: 2-18wks, IQR:4-8wks) and 24 wks (range: 23-35wks, IQR:25-27wks) after diagnosis. We measured neutralizing Ab using an in-house pseudoneutralization assay and anti-N binding Ab using the Abbott Architect assay. Results. Of 32 children enrolled between April 2020 and January 2021, 27 had no underlying immunocompromised state and 25 of these 27 children had symptomatic disease. Ten of 27 had a > 2-fold decrease neutralization titers between 4 and 24wks (most were < 10-fold);12 had < 2-fold change;and 5 had neutralization titers that increased > 2-fold over time (Fig. 1A). All but one of these 27 children had detectable neutralizing activity at 24wks. Anti-N Abs were assessed for 25 children at 4wks and 17 children at 24wks (data pending for 14 samples);all children with paired samples had a > 1.75-fold Abbott index reduction at 24wks, and 5 children had no detectable anti-N Abs by 24wks (Fig. 2A). An additional 5 children with symptomatic disease had complicating immunosuppression or multiple blood transfusions;2 had decreasing neutralizing titers, 2 increased, and 1 had no change (Fig. 1B). Anti-N Abs were undetectable for one child by 24wks (data pending for 4 samples) (Fig. 2B). No participants received COVID-19 vaccine. Conclusion. We show neutralizing Abs wane to a small degree over 24wks post-SARS-CoV-2 infection and remain detectable in most children. In contrast, anti-N Abs decreased, becoming undetectable in some children by 24wks. These findings add to understanding of the natural history of SARS-CoV-2 immunity in children.
ABSTRACT
Purpose/Objective(s): On March 11, 2020, the World Health Organization declared COVID-19 a global pandemic, and on March 16, New York City issued an emergency executive order directing all hospitals to cancel elective surgeries. In the wake of this declaration, non-emergent clinical outpatient care drastically changed. The goal of this study was to understand the impact of the pandemic on treatment of head and neck (H&N) cancers. We hypothesized that the pandemic would lead to difficulties in accessing and delivering cancer care, especially detrimental in the face of curative treatments for H&N cancers. Materials/Methods: In this retrospective review, radiation treatment (RT) parameters for H&N cancer patients treated at our institution were extracted from March 1 to June 30 of 2019 and 2020. Treatment intent, stage at presentation, time from consultation to start date, and total duration of radiation treatment were compared by year. Results were summarized by month of consult and year, then analyzed by year using Wilcoxon rank sum and Pearson's Chi-squared tests. Results: From March 1 to June 30, 2020, 53 head and neck cancer patients were seen in consultation, a 29% reduction from 2019. Ultimately, 96% of these patients received treatment. In 2020, 62% of patients were treated with definitive RT compared to 56% in 2019. When compared to 2019, there was a 23% and 60% decrease in the number of consultations during April and May, accordingly, with a recovery of these numbers by June. There was no significant difference between stage at presentation, treatment duration, time to treatment start, or number of cancelled appointments between the two groups. Conclusion: Consistent with a decrease in presentation of many non-COVID related illnesses, there was a parallel decrease in treatment of H&N cancer patients within our radiation oncology practice, one of the largest H&N centers located in the initial national epicenter of the pandemic. An increase in relative definitive RT regimens demonstrates the increased utilization of radiotherapy once surgical procedures were postponed. Our data highlights that despite significant uncertainties and limitations during this period, successful efforts were made to provide high-quality cancer care without delays in treatment. Future efforts should focus on preparing medical centers to ensure the delivery of essential cancer treatments such as radiotherapy and minimize non-traditional treatment strategies, particularly in the setting of curative treatments for H&N cancers.
ABSTRACT
The coronavirus pandemic is a vivid reminder of just how interconnected the world is. Global populations are united by a shared vulnerability to this invisible virus, just as they are by the unseen causes of climate change. COVID-19 and the climate crisis, too, have exposed the deep structural inequities among low-income populations and communities of color, disparities that put these populations at a significantly higher risk to both crises. Today, due to globalization, dense urban population growth, and disruptions in the ecosystem, humans are more vulnerable to viruses than at any other time in history. Through an examination of these intersecting relationships, this chapter looks, first, at the biology of coronaviruses, followed by the ecology of human and animal environments, and, last, at the sociology of health disparities. © 2021 selection and editorial matter, J. Michael Ryan;individual chapters, the contributors.