Genomic surveillance of SARS-CoV-2 using long-range PCR primers.

Introduction: Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence nearly all of the ~30,000 nucleotide SARS-CoV-2 genome, using a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs). Methods: In this study we used a set of seven long-range PCR primer pairs to sequence clinical isolates of SARS-CoV-2 on Oxford Nanopore sequencer. These long-range primers generate seven amplicons approximately 4500 bp that covered whole genome of SARS-CoV-2. One of these regions includes the full-length S-gene by using a set of flanking primers. We also evaluated the performance of these long-range primers with Midnight primers by sequencing 94 clinical isolates in a Nanopore flow cell. Results and discussion: Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias. The key finding of this study is that long range primers can be used in single-molecule sequencing of RNA viruses in surveillance of emerging variants. We also show that by designing primers flanking the S-gene, we can obtain reliable identification of SARS-CoV-2 variants.

SARS-CoV-2 pandemic non-pharmacologic interventions temporally associated with reduced pediatric infections due to Mycoplasma pneumoniae and co-infecting respiratory viruses in Arkansas.

Non-pharmacologic interventions (NPIs), such as universal masking, implemented during the SARS-CoV-2 pandemic have reduced respiratory infections among children. This study evaluated the impact of NPIs on Mycoplasma pneumoniae infections in children, analyzing data from two hospitals in Arkansas and examining age-related differences and co-infections with other respiratory viruses. The study was approved by the Institutional Review Board and included patients (≤18 years) with upper respiratory tract symptoms. Data generated from the FilmArray Respiratory Panel were divided into pre-NPI, NPI, and post-NPI periods for analysis. Overall test positivity rate and positivity rate interval changes were evaluated. Statistical differences were determined by Chi-square (χ2 independence) analysis. A total of 100,077 tests were performed, with a statistical increase in testing volume during the NPI and post-NPI periods. The number of positive M. pneumoniae tests decreased by 77% (77 to 18) during the NPI period, then increased by 50% (18 to 27) during the post-NPI period. Preschool and elementary school age groups had the highest number of positive tests during the study at 59 (48%) and 40 (33%), respectively. Reduced M. pneumoniae infections were consistent across age groups. Co-infections with other respiratory viruses, particularly human rhinovirus/enterovirus, were observed at much lower levels. Pediatric M. pneumoniae infections in Arkansas were temporally associated with implementation and discontinuation of NPIs. Specific viral co-infections still occurred, albeit at lower levels during the SARS-CoV-2 pandemic. Because of the slower growth of this bacterium, we expect M. pneumoniae infections to return to pre-pandemic levels within approximately 2 years. IMPORTANCE: Non-pharmacologic interventions (NPIs) effectively curtailed the spread of SARS-CoV-2 and, fortuitously, many other aerosol-transmitted respiratory pathogens. This study included the largest data set of symptomatic, pediatric patients from within the United States spanning a period from November 2017 through December 2023, and encompassed individuals residing in both rural and urban settings. We observed a strong correlation between the implementation and cessation of NPIs with the rate of respiratory infections due to Mycoplasma pneumoniae and viral co-infections. These infections are returning to baseline levels approximately 2 years following NPI cessation. This observation was not unexpected since the replication time for viruses is exponentially faster than that of bacteria. The resurgence of M. pneumoniae and likely other atypical bacterial pathogens is currently in process. Healthcare providers should strongly consider these pathogens in individuals presenting with respiratory tract illnesses.

SARS-CoV-2 Pandemic Non-Pharmacologic Interventions Temporally Associated with Reduced Pediatric Infections Due to Mycoplasma pneumoniae and Co-Infecting Respiratory Viruses in Arkansas.

Introduction: Non-pharmacologic interventions (NPIs), such as universal masking, implemented during the SARS-CoV-2 pandemic have reduced respiratory infections among children. This study focuses on evaluating the impact of NPIs on Mycoplasma pneumoniae infections in children, analyzing data from two hospitals in Arkansas, and examining age-related differences and coinfections with other viruses. Methods: The study was approved by the Institutional Review Board and included patients aged ≤18 years with upper respiratory tract symptoms. Data from the FilmArray® Respiratory Panel (FARP) were collected and divided into pre-NPI and NPI periods for analysis. Total test positivity rate and interval change in the positivity rate were evaluated. Statistical differences were determined by Chi-square (χ2-independence) analysis. Results: A total of 68,949 tests were performed with a statistical increase in testing during the NPI period. The overall test positivity rate for M. pneumoniae decreased by 74% (0.86% to 0.03%) during the NPI period, and the preschool age group had the highest number of positive tests in the pre- and NPI periods (Pre-NPI: n=40, NPI: n=12 positive tests, p=<0.001). The reduction in M. pneumoniae infections was consistent across age groups. Coinfections with other respiratory viruses, particularly human rhinovirus/enterovirus, were observed at much lower levels. Conclusions: NPIs effectively reduced M. pneumoniae in pediatric patients in Arkansas, and coinfections with specific viruses still occurred, albeit at lower levels during the SARS-CoV-2 pandemic. As NPIs are relaxed and the pandemic ends, we expect M. pneumoniae infections to return to pre-pandemic levels within the next 1-2 years.

Childhood respiratory viral infections and the microbiome.

The human microbiome associated with the respiratory tract is diverse, heterogeneous, and dynamic. The diversity and complexity of the microbiome and the interactions between microorganisms, host cells, and the host immune system are complex and multifactorial. Furthermore, the lymphatics provide a direct highway, the gut-lung axis, for the gut microbiome to affect outcomes related to respiratory disease and the host immune response. Viral infections in the airways can also alter the presence or absence of bacterial species, which might increase the risks for allergies and asthma. Viruses infect the airway epithelium and interact with the host to promote inflammatory responses that can trigger a wheezing illness. This immune response may alter the host's immune response to microbes and allergens, leading to T2 inflammation. However, exposure to specific bacteria may also tailor the host's response long before the virus has infected the airway. The frequency of viral infections, age at infection, sampling season, geographic location, population differences, and preexisting composition of the microbiota have all been linked to changes in microbiota diversity and stability. This review aims to evaluate the current reported evidence for microbiome interactions and the influences that viral infection may have on respiratory and gut microbiota, affecting respiratory outcomes in children.

Genomic Surveillance of SARS-CoV-2 Using Long-Range PCR Primers.

Whole Genome Sequencing (WGS) of the SARS-CoV-2 virus is crucial in the surveillance of the COVID-19 pandemic. Several primer schemes have been developed to sequence the ~30,000 nucleotide SARS-CoV-2 genome that use a multiplex PCR approach to amplify cDNA copies of the viral genomic RNA. Midnight primers and ARTIC V4.1 primers are the most popular primer schemes that can amplify segments of SARS-CoV-2 (400 bp and 1200 bp, respectively) tiled across the viral RNA genome. Mutations within primer binding sites and primer-primer interactions can result in amplicon dropouts and coverage bias, yielding low-quality genomes with 'Ns' inserted in the missing amplicon regions, causing inaccurate lineage assignments, and making it challenging to monitor lineage-specific mutations in Variants of Concern (VoCs). This study uses seven long-range PCR primers with an amplicon size of ~4500 bp to tile across the complete SARS-CoV-2 genome. One of these regions includes the full-length S-gene by using a set of flanking primers. Using a small set of long-range primers to sequence SARS-CoV-2 genomes reduces the possibility of amplicon dropout and coverage bias.

A longitudinal study of SARS-CoV-2 antibody seroprevalence and mitigation behaviors among college students at an Arkansas University.

Objective: Assess university students' SARS-CoV-2 antibody seroprevalence and mitigation behaviors over time. Participants: Randomly selected college students (N = 344) in a predominantly rural Southern state. Methods: Participants provided blood samples and completed self-administered questionnaires at three timepoints over the academic year. Adjusted odds ratios and 95% confidence intervals were estimated from logistic regression analyses. Results: SARS-CoV-2 antibody seroprevalence was 18.2% in September 2020, 13.1% in December, and 45.5% in March 2021 (21% for those with no vaccination history). SARS-CoV-2 antibody seroprevalence was associated with large social gatherings, staying local during the summer break, symptoms of fatigue or rhinitis, Greek affiliation, attending Greek events, employment, and using social media as the primary COVID-19 information source. In March 2021, seroprevalence was associated with receiving at least one dose of a COVID-19 vaccination. Conclusion: SARS-CoV-2 seroprevalence was higher in this population of college students than previous studies. Results can assist leaders in making informed decisions as new variants threaten college campuses.

An ex vivo human precision-cut lung slice platform provides insight into SARS-CoV-2 pathogenesis and antiviral drug efficacy.

COVID-19 has claimed millions of lives since the emergence of SARS-CoV-2, and lung disease appears the primary cause of the death in COVID-19 patients. However, the underlying mechanisms of COVID-19 pathogenesis remain elusive, and there is no existing model where the human disease can be faithfully recapitulated and conditions for the infection process can be experimentally controlled. Herein we report the establishment of an ex vivo human precision-cut lung slice (hPCLS) platform for studying SARS-CoV-2 pathogenicity and innate immune responses, and for evaluating the efficacy of antiviral drugs against SARS-CoV-2. We show that while SARS-CoV-2 continued to replicate during the course of infection of hPCLS, infectious virus production peaked within 2 days, and rapidly declined thereafter. Although most proinflammatory cytokines examined were induced by SARS-CoV-2 infection, the degree of induction and types of cytokines varied significantly among hPCLS from individual donors, reflecting the heterogeneity of human populations. In particular, two cytokines (IP-10 and IL-8) were highly and consistently induced, suggesting a role in the pathogenesis of COVID-19. Histopathological examination revealed focal cytopathic effects late in the infection. Transcriptomic and proteomic analyses identified molecular signatures and cellular pathways that are largely consistent with the progression of COVID-19 in patients. Furthermore, we show that homoharringtonine, a natural plant alkaloid derived from Cephalotoxus fortunei , not only inhibited virus replication but also production of pro-inflammatory cytokines, and ameliorated the histopathological changes of the lungs caused by SARS-CoV-2 infection, demonstrating the usefulness of the hPCLS platform for evaluating antiviral drugs. SIGNIFICANCE: Here we established an ex vivo human precision-cut lung slice platform for assessing SARS-CoV-2 infection, viral replication kinetics, innate immune response, disease progression, and antiviral drugs. Using this platform, we identified early induction of specific cytokines, especially IP-10 and IL-8, as potential predictors for severe COVID-19, and uncovered a hitherto unrecognized phenomenon that while infectious virus disappears at late times of infection, viral RNA persists and lung histopathology commences. This finding may have important clinical implications for both acute and post-acute sequelae of COVID-19. This platform recapitulates some of the characteristics of lung disease observed in severe COVID-19 patients and is therefore a useful platform for understanding mechanisms of SARS-CoV-2 pathogenesis and for evaluating the efficacy of antiviral drugs.

Rural Research Network to engage rural and minority community members in translational research.

Background: To address the high prevalence of health disparities and lack of research opportunities among rural and minority communities, the University of Arkansas for Medical Sciences (UAMS) developed the Rural Research Network in January 2020. Aim: The aim of this report is to describe our process and progress in developing a rural research network. The Rural Research Network provides a platform to expand research participation opportunities to rural Arkansans, many of whom are older adults, low-income individuals, and underrepresented minority populations. Methods: The Rural Research Network leverages existing UAMS Regional Programs family medicine residency clinics within an academic medical center. Results: Since the inception of the Rural Research Network, research infrastructure and processes have been built within the regional sites. Twelve diverse studies have been implemented with recruitment and data collection from 9248 participants, and 32 manuscripts have been published with residents and faculty from the regional sites. Most studies were able to recruit Black/African American participants at or above a representative sample. Conclusions: As the Rural Research Network matures, the types of research will expand in parallel with the health priorities of Arkansas. Relevance to Patients: The Rural Research Network demonstrates how Cancer Institutes and sites funded by a Clinical and Translational Science Award can collaborate to expand research capacity and increase opportunities for research among rural and minority communities.

Younger and Rural Children are More Likely to be Hospitalized for SARS-CoV-2 Infections.

Purpose: To identify characteristics of SARS-CoV-2 infection that are associated with hospitalization in children initially evaluated in a Pediatric Emergency Department (ED). Methods: We identified cases of SARS-CoV-2 positive patients seen in the Arkansas Children's Hospital (ACH) ED or hospitalized between May 27, 2020, and April 28, 2022 using ICD-10 codes within the Pediatric Hospital Information System (PHIS) Database. We compared infection waves for differences in patient characteristics, and used logistic regressions to examine which characteristics led to a higher chance of hospitalization. Findings: We included 681 pre-Delta cases, 673 Delta cases, and 970 Omicron cases. Almost 17% of patients were admitted to the hospital. Compared to Omicron infected children, pre-Delta and Delta infected children were twice as likely to be hospitalized (OR=2.2 and 2.0, respectively; p<0.0001). Infants less than 1 year of age were >3 times as likely to be hospitalized than children ages 5-14 years regardless of wave (OR=3.42; 95%CI=2.36-4.94). Rural children were almost 3 times as likely than urban children to be hospitalized across all waves (OR=2.73; 95%CI=1.97-3.78). Finally, those with a complex condition had nearly a 15-fold increase in odds of admission (OR=14.6; 95%CI=10.6-20.0). Conclusions: Children diagnosed during the pre-Delta or Delta waves were more likely to be hospitalized than those diagnosed during the Omicron wave. Younger and rural patients were more likely to be hospitalized regardless of wave. We suspect lower vaccination rates and larger distances from medical care influenced higher hospitalization rates.

T-helper 2 mechanisms involved in human rhinovirus infections and asthma.

Human rhinovirus (HRV) is the most common causative agent for the common cold and its respiratory symptoms. For those with asthma, cystic fibrosis, or chronic obstructive pulmonary disease, HRVs can lead to severe and, at times, fatal complications. Furthermore, an array of innate and adaptive host immune responses leads to varying outcomes ranging from subclinical to severe. In this review, we discuss the viral pathogenesis and host immune responses associated with this virus. Specifically, we focus on the immune responses that might skew a T-helper type 2 response, including alarmins, in those with allergic asthma. We also discuss the role of a poor innate immune response with interferons. Finally, we consider therapeutic options for HRV-associated exacerbations of asthma, including biologics and intranasal sprays on the basis of the current literature.

Viral infection and allergy status impact severity of asthma symptoms in children with asthma exacerbations.

BACKGROUND: Although viral infection is known to be associated with asthma exacerbations, prior research has not identified reliable predictors of acute symptom severity in virus-related asthma exacerbations (VRAEs). OBJECTIVE: To determine the effect of asthma control and viral infection on the severity of current illness and evaluate biomarkers related to acute symptoms during asthma exacerbations. METHODS: We prospectively enrolled 120 children with physician-diagnosed asthma and current wheezing who presented to Arkansas Children's Hospital emergency department. The asthma control test (ACT) stratified controlled (ACT > 19) and uncontrolled (ACT ≤ 19) asthma, whereas pediatric respiratory symptom scores evaluated symptoms. Nasopharyngeal swabs were obtained for viral analysis, and inflammatory mediators were evaluated by nasal filter paper and Luminex assays. RESULTS: There were 33 children with controlled asthma and 87 children with uncontrolled asthma. In those with uncontrolled asthma, 77% were infected with viruses during VRAE compared with 58% of those with controlled asthma. Uncontrolled subjects with VRAE had more acute symptoms compared with the controlled subjects with VRAE or uncontrolled subjects without a virus. The uncontrolled subjects with VRAE and allergy had the highest acute symptom scores (3.363 point pediatric respiratory symptom; P = .04). Children with asthma with higher symptom scores had more periostin (P = .02). CONCLUSION: Detection of respiratory viruses is frequent in those with uncontrolled asthma. Uncontrolled subjects with viruses have more acute symptoms during exacerbations, especially in those with allergy. Periostin was highest in subjects with the most acute symptoms, regardless of control status. Taken together, these data imply synergy between viral infection and allergy in subjects with uncontrolled asthma when considering acute asthma symptoms and nasal inflammation during an exacerbation of asthma.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies" (https://www.cancer.gov/research/key-initiatives/covid-19/coronavirus-research-initiatives/serological-sciences-network). SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serology standard reference material and first WHO international standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. SeroNet institutions reported development of a total of 27 enzyme-linked immunosorbent assay (ELISA) methods, 13 multiplex assays, and 9 neutralization assays and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. In conclusion, SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons. IMPORTANCE SeroNet institutions have developed or implemented 61 diverse COVID-19 serological assays and are collaboratively working to harmonize these assays using reference materials to establish standardized reporting units. This will facilitate clinical interpretation of serology results and cross-comparison of research data.

Temporal Variations in Seroprevalence of Severe Acute Respiratory Syndrome Coronavirus 2 Infections by Race and Ethnicity in Arkansas.

Background: The aim of this study was to estimate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection rates in the small rural state of Arkansas, using SARS-CoV-2 antibody prevalence as an indicator of infection. Methods: We collected residual serum samples from adult outpatients seen at hospitals or clinics in Arkansas for non-coronavirus disease 2019 (COVID-19)-related reasons. A total of 5804 samples were identified over 3 time periods: 15 August-5 September 2020 (time period 1), 12 September-24 October 2020 (time period 2), and 7 November-19 December 2020 (time period 3). Results: The age-, sex-, race-, and ethnicity-standardized SARS-CoV-2 seroprevalence during each period, from 2.6% in time period 1 to 4.1% in time period 2 and 7.4% in time period 3. No statistically significant difference in seroprevalence was found based on age, sex, or residence (urban vs rural). However, we found higher seroprevalence rates in each time period for Hispanics (17.6%, 20.6%, and 23.4%, respectively) and non-Hispanic Blacks (4.8%, 5.4%, and 8.9%, respectively) relative to non-Hispanic Whites (1.1%, 2.6%, and 5.5%, respectively). Conclusions: Our data imply that the number of Arkansas residents infected with SARS-CoV-2 rose steadily from 2.6% in August to 7.4% in December 2020. There was no statistical difference in seroprevalence between rural and urban locales. Hispanics and Blacks had higher rates of SARS-CoV-2 antibodies than Whites, indicating that SARS-CoV-2 spread disproportionately in racial and ethnic minorities during the first year of the COVID-19 pandemic.

Seroprevalence of SARS-CoV-2 antibodies in front-line pediatric health care workers.

Objective: The goal of this study was to determine the prevalence of SARS-CoV-2 infections in pediatric front-line health care workers (HCWs) using SARS-CoV-2 serum antibodies as an indicator of infection. Methods: In this cross-sectional study, we collected blood samples and survey responses from HCWs in a 38-bed pediatric emergency department. Serum antibodies to SARS-CoV-2 (IgM and/or IgG) were measured using a 2-step enzyme-linked immunosorbent assay (ELISA) to detect antibodies against the Spike protein receptor binding domain (RBD), the ectodomain of Spike (S), and the nucleoprotein (N). Results: We collected survey responses and serum samples from 54 pediatric front-line HCWs from October 2020 through April 2021. Among the 29 unvaccinated HCWs, 4 (13.7%) had antibodies to SARS-CoV-2. For the 25 vaccinated HCWs, 10 (40%) were seropositive; 3 were <10 days from the first vaccine dose and 7 were ≥10 days after the first dose. Two of the 10 seropositive vaccines had a prior positive reverse transcription polymerase chain reaction test. Individuals ≥10 days from receiving the first vaccine dose were 37.5 (95% CI: 3.5-399.3) times more likely to have SARS-CoV-2 antibodies than unvaccinated individuals or those <10 days from first vaccine dose. Conclusions: Evidence of widespread SARS-CoV-2 infections was not found in unvaccinated front-line HCWs from a pediatric ED as of April 2021. Future work will be required to determine the reasons underlying the lower SARS-CoV-2 antibody prevalence compared to adult HCWs.

State-wide random seroprevalence survey of SARS-CoV-2 past infection in a southern US State, 2020.

The purpose of this cross-sectional study was to estimate the proportion of Arkansas residents who were infected with the SARS-CoV-2 virus between May and December 2020 and to assess the determinants of infection. To estimate seroprevalence, a state-wide population-based random-digit dial sample of non-institutionalized adults in Arkansas was surveyed. Exposures were age, sex, race/ethnicity, education, occupation, contact with infected persons, comorbidities, height, and weight. The outcome was past COVID-19 infection measured by serum antibody test. We found a prevalence of 15.1% (95% CI: 11.1%, 20.2%) by December 2020. Seropositivity was significantly elevated among participants who were non-Hispanic Black, Hispanic (prevalence ratio [PRs]:1.4 [95% CI: 0.8, 2.4] and 2.3 [95% CI: 1.3, 4.0], respectively), worked in high-demand essential services (PR: 2.5 [95% CI: 1.5, 4.1]), did not have a college degree (PR: 1.6 [95% CI: 1.0, 2.4]), had an infected household or extra-household contact (PRs: 4.7 [95% CI: 2.1, 10.1] and 2.6 [95% CI: 1.2, 5.7], respectively), and were contacted in November or December (PR: 3.6 [95% CI: 1.9, 6.9]). Our results indicate that by December 2020, one out six persons in Arkansas had a past SARS-CoV-2 infection.

Pediatric SARS-CoV-2 Seroprevalence in Arkansas Over the First Year of the COVID-19 Pandemic.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) seroprevalence studies largely focus on adults, but little is known about spread in children. We determined SARS-CoV-2 seroprevalence in children and adolescents from Arkansas over the first year of the coronavirus disease of 2019 (COVID-19) pandemic. METHODS: We tested remnant serum samples from children ages 1-18 years who visited Arkansas hospitals or clinics for non-COVID-19-related reasons from April 2020 through April 2021 for SARS-CoV-2 antibodies. We used univariable and multivariable regression models to determine the association between seropositivity and participant characteristics. RESULTS: Among 2357 participants, seroprevalence rose from 7.9% in April/May 2020 (95% CI, 4.9-10.9) to 25.0% in April 2021 (95% CI, 21.5-28.5). Hispanic and black children had a higher association with antibody positivity than non-Hispanic and white children, respectively, in multiple sampling periods. CONCLUSIONS: By spring 2021, most children in Arkansas were not infected with SARS-CoV-2. With the emergence of SARS-CoV-2 variants, recognition of long-term effects of COVID-19, and the lack of an authorized pediatric SARS-CoV-2 vaccine at the time, these results highlight the importance of including children in SARS-CoV-2 public health, clinical care, and research strategies.

The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization.

Background: In October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. Methods: To facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. Results: SeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. Conclusions: SeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Lower viral loads in subjects with rhinovirus-challenged allergy despite reduced innate immunity.

BACKGROUND: Viral infections, especially those caused by rhinovirus, are the most common cause of asthma exacerbations. Previous studies have argued that impaired innate antiviral immunity and, as a consequence, more severe infections contribute to these exacerbations. OBJECTIVE: These studies explored the innate immune response in the upper airway of volunteers with allergic rhinitis and asthma in comparison to healthy controls and interrogated how these differences corresponded to severity of infection. METHODS: Volunteers with allergic rhinitis, those with asthma, and those who are healthy were inoculated with rhinovirus A16 and monitored for clinical symptoms. Tissue and nasal wash samples were evaluated for antiviral signature and viral load. RESULTS: Both subjects with allergic rhinitis and asthma were found to have more severe cold symptoms. Subjects with asthma had worsened asthma control and increased bronchial hyperreactivity in the setting of higher fractional exhaled breath nitric oxide and blood eosinophils. These studies confirmed reduced expression of interferons and virus-specific pattern recognition receptors in both cohorts with atopy. Nevertheless, despite this defect in innate immunity, volunteers with allergic rhinitis/asthma had reduced rhinovirus concentrations in comparison to the controls. CONCLUSION: These results confirm that the presence of an allergic inflammatory disorder of the airway is associated with reduced innate immune responsive to rhinovirus infection. Despite this, these volunteers with allergy have reduced viral loads, arguing for the presence of a compensatory mechanism to clear the infection. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02910401.

Development of ACE2 autoantibodies after SARS-CoV-2 infection.

BACKGROUND: Activation of the immune system is implicated in the Post-Acute Sequelae after SARS-CoV-2 infection (PASC) but the mechanisms remain unknown. Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin II (Ang II) resulting in decreased activation of the AT1 receptor and decreased immune system activation. We hypothesized that autoantibodies against ACE2 may develop after SARS-CoV-2 infection, as anti-idiotypic antibodies to anti-spike protein antibodies. METHODS AND FINDINGS: We tested plasma or serum for ACE2 antibodies in 67 patients with known SARS-CoV-2 infection and 13 with no history of infection. None of the 13 patients without history of SARS-CoV-2 infection and 1 of the 20 outpatients that had a positive PCR test for SARS-CoV-2 had levels of ACE2 antibodies above the cutoff threshold. In contrast, 26/32 (81%) in the convalescent group and 14/15 (93%) of patients acutely hospitalized had detectable ACE2 antibodies. Plasma from patients with antibodies against ACE2 had less soluble ACE2 activity in plasma but similar amounts of ACE2 protein compared to patients without ACE2 antibodies. We measured the capacity of the samples to inhibit ACE2 enzyme activity. Addition of plasma from patients with ACE2 antibodies led to decreased activity of an exogenous preparation of ACE2 compared to patients that did not have antibodies. CONCLUSIONS: Many patients with a history of SARS-CoV-2 infection have antibodies specific for ACE2. Patients with ACE2 antibodies have lower activity of soluble ACE2 in plasma. Plasma from these patients also inhibits exogenous ACE2 activity. These findings are consistent with the hypothesis that ACE2 antibodies develop after SARS-CoV-2 infection and decrease ACE2 activity. This could lead to an increase in the abundance of Ang II, which causes a proinflammatory state that triggers symptoms of PASC.