Using a function-first "scout fragment"-based approach to develop allosteric covalent inhibitors of conformationally dynamic helicase mechanoenzymes (preprint)

Helicases, classified into six superfamilies, are mechanoenzymes that utilize energy derived from ATP hydrolysis to remodel DNA and RNA substrates. These enzymes have key roles in diverse cellular processes, such as genome replication and maintenance, ribosome assembly and translation. Helicases with essential functions only in certain cancer cells have been identified and helicases expressed by certain viruses are required for their pathogenicity. As a result, helicases are important targets for chemical probes and therapeutics. However, it has been very challenging to develop selective chemical inhibitors for helicases, enzymes with highly dynamic conformations. We envisioned that electrophilic "scout fragments", which have been used for chemical proteomic based profiling, could be leveraged to develop covalent inhibitors of helicases. We adopted a function-first approach, combining enzymatic assays with enantiomeric probe pairs and mass spectrometry, to develop a covalent inhibitor that selectively targets an allosteric site in SARS-CoV-2 nsp13, a superfamily-1 helicase. Further, we demonstrate that scout fragments inhibit the activity of two human superfamily-2 helicases, BLM and WRN, involved in genome maintenance. Together, our findings suggest a covalent inhibitor discovery approach to target helicases and potentially other conformationally dynamic mechanoenzymes.

Effectiveness of BNT162b2 COVID-19 Vaccination in Children Aged 5-17 Years in the United States (preprint)

Importance: COVID-19 vaccines are authorized for use in children in the United States; real-world assessment of vaccine effectiveness in children is needed. Objective: To estimate the effectiveness of receiving a complete primary series of monovalent BNT162b2 (Pfizer-BioNTech) COVID-19 vaccine in US children. Design: A cohort study of children aged 5--17 years vaccinated with BNT162b2 matched with unvaccinated children. Setting: Participants identified in Optum and CVS Health insurance administrative claims databases were linked with Immunization Information System (IIS) COVID-19 vaccination records from 16 US jurisdictions between December 11, 2020, and May 31, 2022 (end date varied by database and IIS). Participants: Vaccinated children were followed from their first BNT162b2 dose and matched to unvaccinated children on calendar date, US county of residence, and demographic and clinical factors. Censoring occurred if vaccinated children failed to receive a timely dose 2 or if unvaccinated children received any dose. Exposure: BNT162b2 vaccinations were identified using IIS vaccination records and insurance claims. Main Outcomes and Measures: Two COVID-19 outcome definitions were evaluated: COVID-19 diagnosis in any medical setting and COVID-19 diagnosis in hospitals/emergency departments (EDs). Propensity score-weighted hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated with Cox proportional hazards models, and vaccine effectiveness (VE) was estimated as 1 minus HR. VE was estimated overall, within age subgroups, and within variant-specific eras. Sensitivity, negative control, and quantitative bias analyses evaluated various potential biases. Results: There were 453,655 eligible vaccinated children one-to-one matched to unvaccinated comparators (mean age 12 years; 50% female). COVID-19 hospitalizations/ED visits were rare in children, regardless of vaccination status (Optum, 41.2 per 10,000 person- years; CVS Health, 44.1 per 10,000 person- years). Overall, vaccination was associated with reduced incidence of any medically diagnosed COVID-19 (meta-analyzed VE = 38% [95% CI, 36%-40%]) and hospital/ED-diagnosed COVID-19 (meta-analyzed VE = 61% [95% CI, 56%-65%]). VE estimates were lowest among children 5--11 years and during the omicron variant era. Conclusions and Relevance: Receipt of a complete BNT162b2 vaccine primary series was associated with overall reduced medically diagnosed COVID-19 and hospital/ED-diagnosed COVID-19 in children; observed VE estimates differed by age group and variant era.

Cohort-specific serological recognition of SARS-CoV-2 variant RBD antigens (preprint)

Background Estimating the response of different cohorts (e.g. vaccinated or critically ill) to new SARS-CoV-2 variants is important to customize measures of control. Thus, our goal was to evaluate binding of antibodies from sera of infected and vaccinated people to different antigens expressed by SARS-CoV-2 variants. Methods We compared sera from vaccinated donors with sera from four patient/donor cohorts: critically ill patients admitted to an intensive care unit (split in sera collected between 2 and 7 days after admission and more than ten days later), a NIBSC/WHO reference panel of SARS-CoV-2 positive individuals, and ambulatory or hospitalized (but not critically ill) positive donors. Samples were tested with an anti-SARS-CoV-2 IgG serological assay designed with microplates coated with a SARS-CoV-2 RBD recombinant antigen. The same sample sets were also tested with microplates coated with antigens harbouring RBD mutations present in eleven of the most widespread variants. Results Sera from vaccinated individuals exhibited higher antibody binding (P<0.001) than sera from infected (but not critically ill) individuals when tested against the WT and each of 11 variants’ RBD. The optical density generated by sera from non-critically ill convalescence individuals upon binding to variant’s antigens was different (P<0.05) from that of the WT in some variants—noteworthy, Beta, Gamma, Delta, and Delta Plus variants. Conclusions Understanding differences in binding and neutralizing antibody titers against WT vs variant RBD antigens from different donor cohorts can help design variant-specific immunoassays and complement other diagnostic and clinical data to evaluate the epidemiology of new variants.

Impact of the COVID-19 Stay-at-Home Order on Diet and Health in Community-Dwelling Older Adults With Obesity

Profound restrictions were placed on previously free-living older adults due to mandatory stay-at-home orders for Covid-19. Recognizing the potential for worsening health and heightened risk of Covid-19 complications with older age and obesity, we conducted a survey to assess the impact of stay-at-home requirements on diet, health/social behaviors, and food security in 58 older adults (age=70.8±6.2, 55% Black, 93% female) who had participated in past obesity-reduction trials. A 71-item questionnaire was administered by phone and included demographics, health, lifestyle and dietary habits, food attitudes, and food security questions. Results showed indicators of heightened health risk, including health care appointments either delayed/cancelled (69%) and self-reported weight gain (62%). Of those with weight gain, 22% reported a gain of >10 pounds (33% gained 5-10 pounds and 7% < 5 pounds). Increased food intake was reported by 67% and 45% felt their eating patterns were less healthy due to increased snacking (71%) and consumption of sweets (41%). Food access and isolation were also an issue, as 51% were concerned about leaving the house for food and 81% reported eating alone. While some positive behaviors were reported (new ways to access food and health information, more reported cooking at home), the majority of findings indicated increased risk of obesity and its complications. The findings emphasize the need for effective lifestyle interventions that can be delivered remotely to high-risk older adults;this would benefit those presently self-restricted for Covid-19 concerns as well as other isolated older adults who need better access to individualized interventions.

The COVIDome Explorer Researcher Portal (preprint)

COVID-19 pathology involves dysregulation of diverse molecular, cellular, and physiological processes. In order to expedite integrated and collaborative COVID-19 research, we completed multi-omics analysis of hospitalized COVID-19 patients including matched analysis of the whole blood transcriptome, plasma proteomics with two complementary platforms, cytokine profiling, plasma and red blood cell metabolomics, deep immune cell phenotyping by mass cytometry, and clinical data annotation. We refer to this multidimensional dataset as the COVIDome. We then created the COVIDome Explorer, an online researcher portal where the data can be analyzed and visualized in real time. We illustrate here the use of the COVIDome dataset through a multi-omics analysis of biosignatures associated with C-reactive protein (CRP), an established marker of poor prognosis in COVID-19, revealing associations between CRP levels and damage-associated molecular patterns, depletion of protective serpins, and mitochondrial metabolism dysregulation. We expect that the COVIDome Explorer will rapidly accelerate data sharing, hypothesis testing, and discoveries worldwide.Funding: This work was supported by NIH grants R01AI150305, 3R01AI150305-01S1, R01AI145988, UL1TR002535, 3UL1TR002535-03S2, R01HL146442, R01HL149714, R01HL148151, R21HL150032, P30CA046934, R35GM124939 and RM1GM131968, as well as grants from the Boettcher Foundation and Fast Grants. Additional support was received from Chancellor’s Discovery Innovation Fund at the CU Anschutz Medical Campus, the Global Down Syndrome Foundation, the Anna and John J. Sie Foundation, and Lyda Hill Philanthropies.Conflict of Interest: KDS and JME are co-inventors on two patents related to JAK inhibition in COVID-19; JME serves in the COVID Development Advisory Board for Elly Lilly and has provided consulting services to Gilead Sciences Inc. JME serves on the Cell Reports Advisory Board.

COVID-19 Neuropathology at Columbia University Irving Medical Center/New York Presbyterian Hospital (preprint)

Many patients with SARS-CoV-2 infection develop neurological signs and symptoms, though, to date, little evidence exists that primary infection of the brain is a significant contributing factor. We present the clinical, neuropathological, and molecular findings of 41 consecutive patients with SARS-CoV-2 infections who died and underwent autopsy in our medical center. The mean age was 74 years (38-97 years), 27 patients (66%) were male and 34 (83%) were of Hispanic/Latinx ethnicity. Twenty-four patients (59%) were admitted to the intensive care unit (ICU). Hospital-associated complications were common, including 8 (20%) with deep vein thrombosis/pulmonary embolism (DVT/PE), 7 (17%) patients with acute kidney injury requiring dialysis, and 10 (24%) with positive blood cultures during admission. Eight (20%) patients died within 24 hours of hospital admission, while 11 (27%) died more than 4 weeks after hospital admission. Neuropathological examination of 20-30 areas from each brain revealed hypoxic/ischemic changes in all brains, both global and focal; large and small infarcts, many of which appeared hemorrhagic; and microglial activation with microglial nodules accompanied by neuronophagia, most prominently in the brainstem. We observed sparse T lymphocyte accumulation in either perivascular regions or in the brain parenchyma. Many brains contained atherosclerosis of large arteries and arteriolosclerosis, though none had evidence of vasculitis. Eighteen (44%) contained pathologies of neurodegenerative diseases, not unexpected given the age range of our patients. We examined multiple fresh frozen and fixed tissues from 28 brains for the presence of viral RNA and protein, using quantitative reverse-transcriptase PCR (qRT- PCR), RNAscope, and immunocytochemistry with primers, probes, and antibodies directed against the spike and nucleocapsid regions. qRT-PCR revealed low to very low, but detectable, viral RNA levels in the majority of brains, although they were far lower than those in nasal epithelia. RNAscope and immunocytochemistry failed to detect viral RNA or protein in brains. Our findings indicate that the levels of detectable virus in COVID-19 brains are very low and do not correlate with the histopathological alterations. These findings suggest that microglial activation, microglial nodules and neuronophagia, observed in the majority of brains, do not result from direct viral infection of brain parenchyma, but rather likely from systemic inflammation, perhaps with synergistic contribution from hypoxia/ischemia. Further studies are needed to define whether these pathologies, if present in patients who survive COVID-19, might contribute to chronic neurological problems.

The COVIDome Explorer Researcher Portal (preprint)

SUMMARY COVID-19 pathology involves dysregulation of diverse molecular, cellular, and physiological processes. In order to expedite integrated and collaborative COVID-19 research, we completed multi-omics analysis of hospitalized COVID-19 patients including matched analysis of the whole blood transcriptome, plasma proteomics with two complementary platforms, cytokine profiling, plasma and red blood cell metabolomics, deep immune cell phenotyping by mass cytometry, and clinical data annotation. We refer to this multidimensional dataset as the COVIDome. We then created the COVIDome Explorer, an online researcher portal where the data can be analyzed and visualized in real time. We illustrate here the use of the COVIDome dataset through a multi-omics analysis of biosignatures associated with C-reactive protein (CRP), an established marker of poor prognosis in COVID-19, revealing associations between CRP levels and damage-associated molecular patterns, depletion of protective serpins, and mitochondrial metabolism dysregulation. We expect that the COVIDome Explorer will rapidly accelerate data sharing, hypothesis testing, and discoveries worldwide.

Transcriptional Profiling of Leukocytes in Critically Ill COVID19 Patients: Implications for Interferon Response and Coagulation (preprint)

Background: COVID19 is caused by the SARS-CoV-2 virus and has been associated with severe inflammation leading to organ dysfunction and mortality. Our aim was to profile the transcriptome in leukocytes from critically ill patients positive for COVID19 compared to those negative for COVID19 to better understand the COVID19 associated host response. For these studies, all patients admitted to our tertiary care intensive care unit (ICU) suspected of being infected with SARS-CoV-2, using standardized hospital screening methodologies, had blood samples collected at the time of admission to the ICU. Transcriptome profiling of leukocytes via ribonucleic acid sequencing (RNAseq) was then performed and differentially expressed genes as well as significantly enriched gene sets were identified. Results: : We enrolled seven COVID19+ (PCR positive, 2 SARS-CoV-2 genes) and seven age- and sex-matched COVID19- (PCR negative) control ICU patients. Cohorts were well-balanced with the exception that COVID19- patients had significantly higher total white blood cell counts and circulating neutrophils and COVID19+ patients were more likely to suffer bilateral pneumonia. The mortality rate for this cohort of COVID19+ ICU patients was 29%. As indicated by both single-gene based and gene set (GSEA) approaches, the major disease-specific transcriptional responses of leukocytes in critically ill COVID19+ ICU patients were: (i) a robust overrepresentation of interferon related gene expression; (ii) a marked decrease in the transcriptional level of genes contributing to general protein synthesis and bioenergy metabolism; and (iii) the dysregulated expression of genes associated with coagulation, platelet function, complement activation, and tumour necrosis factor/interleukin 6 signalling. Conclusions: : Our findings demonstrate that critically ill COVID19+ patients on day 1 of admission to the ICU display a unique leukocyte transcriptional profile that distinguishes them from COVID19- patients, providing guidance for future targeted studies exploring novel prognostic and therapeutic aspects of COVID19.