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Some studies suggest that recent common coronavirus (CCV) infections are associated with reduced COVID-19 severity upon SARS-CoV-2 infection. We completed serological assays using samples collected from health care workers to identify antibody types associated with SARS-CoV-2 protection and COVID-19 symptom duration. Rare SARS-CoV-2 cross-reactive antibodies elicited by past CCV infections were not associated with protection; however, the duration of symptoms following SARS-CoV-2 infections was significantly reduced in individuals with higher common betacoronavirus (βCoV) antibody titers. Since antibody titers decline over time after CCV infections, individuals in our cohort with higher βCoV antibody titers were more likely recently infected with common βCoVs compared with individuals with lower antibody titers. Therefore, our data suggest that recent βCoV infections potentially limit the duration of symptoms following SARS-CoV-2 infections through mechanisms that do not involve cross-reactive antibodies. Our data are consistent with the emerging hypothesis that cellular immune responses elicited by recent common βCoV infections transiently reduce symptom duration following SARS-CoV-2 infections.

Influenza is a significant cause of morbidity and mortality worldwide. Here we show changes in the abundance and activation states of more than 50 immune cell subsets in 35 individuals over 11 time points during human A/California/2009 (H1N1) virus challenge monitored using mass cytometry along with other clinical assessments. Peak change in monocyte, B cell, and T cell subset frequencies coincided with peak virus shedding, followed by marked activation of T and NK cells. Results led to the identification of CD38 as a critical regulator of plasmacytoid dendritic cell function in response to influenza virus. Machine learning using study-derived clinical parameters and single-cell data effectively classified and predicted susceptibility to infection. The coordinated immune cell dynamics defined in this study provide a framework for identifying novel correlates of protection in the evaluation of future influenza therapeutics.

To describe the cumulative seroprevalence of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) antibodies during the coronavirus disease 2019 (COVID-19) pandemic among employees of a large pediatric healthcare system. Prospective observational cohort study open to adult employees at the Children's Hospital of Philadelphia, conducted April 20-December 17, 2020. Employees were recruited starting with high-risk exposure groups, utilizing e-mails, flyers, and announcements at virtual town hall meetings. At baseline, 1 month, 2 months, and 6 months, participants reported occupational and community exposures and gave a blood sample for SARS-CoV-2 antibody measurement by enzyme-linked immunosorbent assays (ELISAs). A post hoc Cox proportional hazards regression model was performed to identify factors associated with increased risk for seropositivity. In total, 1,740 employees were enrolled. At 6 months, the cumulative seroprevalence was 5.3%, which was below estimated community point seroprevalence. Seroprevalence was 5.8% among employees who provided direct care and was 3.4% among employees who did not perform direct patient care. Most participants who were seropositive at baseline remained positive at follow-up assessments. In a post hoc analysis, direct patient care (hazard ratio [HR], 1.95; 95% confidence interval [CI], 1.03-3.68), Black race (HR, 2.70; 95% CI, 1.24-5.87), and exposure to a confirmed case in a nonhealthcare setting (HR, 4.32; 95% CI, 2.71-6.88) were associated with statistically significant increased risk for seropositivity. Employee SARS-CoV-2 seroprevalence rates remained below the point-prevalence rates of the surrounding community. Provision of direct patient care, Black race, and exposure to a confirmed case in a nonhealthcare setting conferred increased risk. These data can inform occupational protection measures to maximize protection of employees within the workplace during future COVID-19 waves or other epidemics.

An H1N1 influenza virus caused a pandemic in 2009 and descendants of this virus continue to circulate seasonally in humans. Upon infection with the 2009 H1N1 pandemic strain (pH1N1), many humans produced antibodies against epitopes in the hemagglutinin (HA) stalk. HA stalk-focused antibody responses were common among pH1N1-infected individuals because HA stalk epitopes were conserved between the pH1N1 strain and previously circulating H1N1 strains. Here, we completed a series of experiments to determine if the pH1N1 HA stalk has acquired substitutions since 2009 that prevent the binding of human antibodies. We identified several amino acid substitutions that have accrued in the pH1N1 HA stalk from 2009-2019. We completed enzyme-linked immunosorbent assays, absorption-based binding assays, and surface plasmon resonance experiments to determine if these substitutions affect antibody binding. Using sera collected from 230 humans (aged 21-80 years), we found that pH1N1 HA stalk substitutions that have emerged since 2009 do not affect antibody binding. Our data suggest that the HA stalk domain of pH1N1 viruses remained antigenically stable after circulating in humans for a decade.

Seasonal influenza viruses are a major cause of human disease worldwide. Most neutralizing antibodies (Abs) elicited by influenza viruses target the head domain of the hemagglutinin (HA) protein. Anti-HA head Abs can be highly potent, but they have limited breadth since the HA head is variable. There is great interest in developing new universal immunization strategies that elicit broadly neutralizing Abs against conserved regions of HA, such as the stalk domain. Although HA stalk Abs can provide protection in animal models, it is unknown if they are present at sufficient levels in humans to provide protection against naturally-acquired influenza virus infections. Here, we quantified H1N1 HA head and stalk-specific Abs in 179 adults hospitalized during the 2015-2016 influenza virus season. We found that HA head Abs, as measured by hemagglutinin-inhibition (HAI) assays, were associated with protection against naturally-acquired H1N1 infection. HA stalk-specific serum total IgG titers were also associated with protection, but this association was slightly attenuated and not statistically significant after adjustment for HA head-specific Ab titers. We found higher titers of HA stalk-specific IgG1 and IgA Abs in sera from uninfected participants than from infected participants; however, we found no difference in sera in vitro antibody dependent cellular cytotoxicity activity. In passive transfer experiments, sera from participants with high HAI activity efficiently protected mice, while sera with low HAI activity protected mice to a lower extent. Our data suggest that human HA head and stalk Abs both contribute to protection against H1N1 infection.

Recent progress in targeting KRAS G12C has provided both insight and inspiration for targeting alternative KRAS mutants. In this study, we evaluated the mechanism of action and anti-tumor efficacy of MRTX1133, a potent, selective and non-covalent KRAS G12D inhibitor. MRTX1133 demonstrated a high-affinity interaction with GDP-loaded KRAS G12D with K D and IC 50 values of ~0.2 pM and <2 nM, respectively, and ~700-fold selectivity for binding to KRAS G12D as compared to KRAS WT . MRTX1133 also demonstrated potent inhibition of activated KRAS G12D based on biochemical and co-crystal structural analyses. MRTX1133 inhibited ERK1/2 phosphorylation and cell viability in KRAS G12D -mutant cell lines, with median IC 50 values of ~5 nM, and demonstrated >1,000-fold selectivity compared to KRAS WT cell lines. MRTX1133 exhibited dose-dependent inhibition of KRAS-mediated signal transduction and marked tumor regression (≥30%) in a subset of KRAS G12D -mutant cell-line-derived and patient-derived xenograft models, including eight of 11 (73%) pancreatic ductal adenocarcinoma (PDAC) models. Pharmacological and CRISPR-based screens demonstrated that co-targeting KRAS G12D with putative feedback or bypass pathways, including EGFR or PI3Kα, led to enhanced anti-tumor activity. Together, these data indicate the feasibility of selectively targeting KRAS mutants with non-covalent, high-affinity small molecules and illustrate the therapeutic susceptibility and broad dependence of KRAS G12D mutation-positive tumors on mutant KRAS for tumor cell growth and survival. A potent and selective inhibitor of KRAS G12D , the most common mutant form of the KRAS oncoprotein, has anti-tumor efficacy in multiple pre-clinical cancer models, opening the possibility to therapeutically target this highly prevalent oncogenic driver.

Surveillance of non-ventilator-associated hospital-acquired pneumonia (NV-HAP) is complicated by subjectivity and variability in diagnosing pneumonia. We compared a fully automatable surveillance definition using routine electronic health record data to manual determinations of NV-HAP according to surveillance criteria and clinical diagnoses. We retrospectively applied an electronic surveillance definition for NV-HAP to all adults admitted to Veterans' Affairs (VA) hospitals from January 1, 2015, to November 30, 2020. We randomly selected 250 hospitalizations meeting NV-HAP surveillance criteria for independent review by 2 clinicians and calculated the percent of hospitalizations with (1) clinical deterioration, (2) CDC National Healthcare Safety Network (CDC-NHSN) criteria, (3) NV-HAP according to a reviewer, (4) NV-HAP according to a treating clinician, (5) pneumonia diagnosis in discharge summary; and (6) discharge diagnosis codes for HAP. We assessed interrater reliability by calculating simple agreement and the Cohen κ (kappa). Among 3.1 million hospitalizations, 14,023 met NV-HAP electronic surveillance criteria. Among reviewed cases, 98% had a confirmed clinical deterioration; 67% met CDC-NHSN criteria; 71% had NV-HAP according to a reviewer; 60% had NV-HAP according to a treating clinician; 49% had a discharge summary diagnosis of pneumonia; and 82% had NV-HAP according to any definition according to at least 1 reviewer. Only 8% had diagnosis codes for HAP. Interrater agreement was 75% (κ = 0.50) for CDC-NHSN criteria and 78% (κ = 0.55) for reviewer diagnosis of NV-HAP. Electronic NV-HAP surveillance criteria correlated moderately with existing manual surveillance criteria. Reviewer variability for all manual assessments was high. Electronic surveillance using clinical data may therefore allow for more consistent and efficient surveillance with similar accuracy compared to manual assessments or diagnosis codes.

Age is the largest risk factor for Alzheimer's disease (AD) and contributes to cognitive impairment in otherwise healthy individuals. Thus, it is critical that we better understand the risk aging presents to vulnerable regions of the brain and carefully design therapeutics to address those effects. In this study we examined age-related changes in cAMP-regulatory protein, phosphodiesterase 4D (PDE4D). Inhibition of PDE4D is currently under investigation as a therapeutic target for AD based on memory-enhancing effects in rodent hippocampus. Therefore, it is important to understand the role of PDE4D in brain regions particularly vulnerable to disease such as the frontal association cortex (FC), where cAMP signaling can impair working memory via opening of potassium channels. We found that PDE4D protein level was decreased in the FC of both moderately and extremely aged rats, and that PDE4D level was correlated with performance on a FC-dependent working memory task. In extremely aged rats, PDE4D was also inversely correlated with levels of phosphorylated tau at serine 214 (S214), a site phosphorylated by protein kinase A. studies of the PDE4D inhibitor, GEBR-7b, further illustrated that inhibition of PDE4D activity enhanced phosphorylation of tau. pS214-tau phosphorylation is associated with early AD tau pathology, promotes tau dissociation from microtubules and primes subsequent tau hyperphosphorylation at other critical AD-related sites. Age-related loss of PDE4D may thus contribute to the specific vulnerability of the FC to degeneration in AD, and play a critical role in normal cAMP regulation, cautioning against the use of pan-PDE4D inhibitors as therapeutics.

The Poxviridae family members vaccinia and variola virus enter mammalian cells, replicate outside the nucleus and produce virions that travel to the cell surface along microtubules, fuse with the plasma membrane and egress from infected cells toward apposing cells on actin-filled membranous protrusions. We show that cell-associated enveloped virions (CEV) use Abl- and Src-family tyrosine kinases for actin motility, and that these kinases act in a redundant fashion, perhaps permitting motility in a greater range of cell types. Additionally, release of CEV from the cell requires Abl- but not Src-family tyrosine kinases, and is blocked by STI-571 (Gleevec), an Abl-family kinase inhibitor used to treat chronic myelogenous leukemia in humans. Finally, we show that STI-571 reduces viral dissemination by five orders of magnitude and promotes survival in infected mice, suggesting possible use for this drug in treating smallpox or complications associated with vaccination. This therapeutic approach may prove generally efficacious in treating microbial infections that rely on host tyrosine kinases, and, because the drug targets host but not viral molecules, this strategy is much less likely to engender resistance compared to conventional antimicrobial therapies.

ObjectiveExtremely preterm (EP) impairment rates are likely underestimated using the Bayley III norm-based thresholds scores and may be better assessed relative to concurrent healthy term reference (TR) infants born in the same hospital.Study designBlinded, certified examiners in the Neonatal Research Network (NRN) evaluated EP survivors and a sample of healthy TR infants recruited near the 2-year assessment age.ResultsWe assessed 1452 EP infants and 183 TR infants. TR-based thresholds showed higher overall EP impairment than Bayley norm-based thresholds (O.R. = 1.86; [95% CI 1.56–2.23], especially for severe impairment (36% vs. 24%; p ≤ 0.001).Difficulty recruiting TR patients at 2 years extended the study by 14 months and affected their demographics.ConclusionImpairment rates among EP infants appear to be substantially underestimated from Bayley III norms. These rates may be best assessed by comparison with healthy term infants followed with minimal attrition from birth in the same centers.ClinicalTrials.gov IDTerm Reference (under the Generic Database Study): NCT00063063