SARS-CoV-2 Serosurveys: How antigen, isotype and threshold choices affect the outcome.

BACKGROUND: Evaluating the performance of SARS-CoV-2 serological assays and clearly articulating the utility of selected antigen, isotypes and thresholds is crucial to understanding the prevalence of infection within selected communities. METHODS: This cross-sectional study, implemented in 2020, screened PCR-confirmed COVID-19 patients (n = 86), banked pre-pandemic and negative donors (n = 96), health care workers and family members (n = 552), and university employees (n = 327) for anti-SARS-CoV-2 receptor-binding domain (RBD), trimeric spike protein (S), and nucleocapsid protein (N) IgG and IgA antibodies with a laboratory developed Enzyme-Linked Immunosorbent Assay (ELISA) and tested how antigen, isotype and threshold choices affected the seroprevalence. The following threshold methods were evaluated: (i) mean + 3 standard deviations of the negative controls; (ii) 100% specificity for each antigen/isotype combination; and (iii) the maximal Youden index. RESULTS: We found vastly different seroprevalence estimates depending on selected antigens, isotypes and the applied threshold method, ranging from 0.0% to 85.4% . Subsequently, we maximized specificity and reported a seroprevalence, based on more than one antigen, ranging from 9.3% to 25.9%. CONCLUSIONS: This study revealed the importance of evaluating serosurvey tools for antigen, isotype, and threshold-specific sensitivity and specificity, in order to interpret qualitative serosurvey outcomes reliably and consistently across studies.

Investigating demographic differences in patients' decisions to consent to COVID-19 research.

OBJECTIVE: COVID-19 disease severely impacted pregnant persons, resulting in a significant increase in poor maternal health outcomes, with a disproportionate impact on minority populations and individuals with low socioeconomic status. We sought to determine demographic differences between birthing parents with SARS-CoV-2 infections who consented to research study participation versus those who declined. By analyzing demographic differences, we are able to ensure the generalizability of study outcomes and to aid in future prospective research design, with the ultimate goal of recognizing and ameliorating research disparities. METHODS: We conducted a secondary analysis to investigate demographic differences in patients who consented to versus declined study participation, in an effort to confirm the external validity of the study results and ensure minority populations most affected by SARS-CoV-2 infection were accurately represented. An IRB waiver was obtained to conduct retrospective chart review for demographic data collection of all patients approached for the COVID-19 Analysis on Perinatal Specimens Related to ExpoSure (CARES) research study. Pregnant patients with SARS-CoV-2 infection were identified at a single hospital center and approached either in person or via phone, with a translator if primary language listed as non-English. Demographic variables including race, ethnicity, primary language, and insurance type were obtained from the electronic medical record and analyzed via Chi-square to determine significant differences between individuals who consented to participation and those who declined participation. RESULTS: One hundred and fifty-eight pregnant patients with SARS-CoV-2 infection were approached for CARES study participation. Eighty-nine patients consented to study participation, while 69 declined study participation. A retrospective chart review was conducted on all 158 patients. Patients who identified as Black race or non-White race were more likely to decline participation (23.2%, p = .031, 68.1%, p = .026), compared to patients who identified as White (31.9%) (Table 1). Patients with public insurance were also more likely to decline study participation (72.5%, p = .049) compared to those with private insurance (27.5%). There was no significant difference between primary language spoken or ethnicity in patients who participated or declined. There was no difference in study participation between patients who identified as Asian race or Other race, compared to patients who identified as White race. CONCLUSIONS: We found significant differences in race and insurance type between pregnant patients with SARS-CoV-2 infection who consented versus declined research study participation. Our study showed that patients who identify as Black race or have public insurance are less likely to consent to research study participation. However, when demographics of consented patients are compared to county, state, and national demographics of female patients age 18-49 with confirmed SARS-CoV-2 infection obtained from a dataset collected by the Center for Disease Control and Prevention (CDC), there was no significant difference between race representation of patients who consented to study participation. This suggests that though the external validity of the CARES study is confirmed, more efforts need to be made to address racial and socioeconomic disparities in research participation.

Mission, Organization, and Future Direction of the Serological Sciences Network for COVID-19 (SeroNet) Epidemiologic Cohort Studies.

Background: Global efforts are needed to elucidate the epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the underlying cause of coronavirus disease 2019 (COVID-19), including seroprevalence, risk factors, and long-term sequelae, as well as immune responses after vaccination across populations and the social dimensions of prevention and treatment strategies. Methods: In the United States, the National Cancer Institute in partnership with the National Institute of Allergy and Infectious Diseases, established the SARS-CoV-2 Serological Sciences Network (SeroNet) as the nation's largest coordinated effort to study coronavirus disease 2019. The network comprises multidisciplinary researchers bridging gaps and fostering collaborations among immunologists, epidemiologists, virologists, clinicians and clinical laboratories, social and behavioral scientists, policymakers, data scientists, and community members. In total, 49 institutions form the SeroNet consortium to study individuals with cancer, autoimmune disease, inflammatory bowel diseases, cardiovascular diseases, human immunodeficiency virus, transplant recipients, as well as otherwise healthy pregnant women, children, college students, and high-risk occupational workers (including healthcare workers and first responders). Results: Several studies focus on underrepresented populations, including ethnic minorities and rural communities. To support integrative data analyses across SeroNet studies, efforts are underway to define common data elements for standardized serology measurements, cellular and molecular assays, self-reported data, treatment, and clinical outcomes. Conclusions: In this paper, we discuss the overarching framework for SeroNet epidemiology studies, critical research questions under investigation, and data accessibility for the worldwide scientific community. Lessons learned will help inform preparedness and responsiveness to future emerging diseases.

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.

Development of flow cytometry-based assays to assess the ability of antibodies to bind to SARS-CoV-2-infected and spike-transfected cells and mediate NK cell degranulation.

Since the beginning of the SARS-CoV-2 pandemic, antibody responses and antibody effector functions targeting SARS-CoV-2-infected cells have been understudied. Consequently, the role of these types of antibodies in SARS-CoV-2 disease (COVID-19) and immunity is still undetermined. To provide tools to study these responses, we used plasma from SARS-CoV-2-infected individuals (n = 50) and SARS-CoV-2 naive healthy controls (n = 20) to develop four specific and reproducible flow cytometry-based assays: (i) two assessing antibody binding to, and antibody-mediated NK cell degranulation against, SARS-CoV-2-infected cells and (ii) two assessing antibody binding to, and antibody-mediated NK cell degranulation against, SARS-CoV-2 Spike-transfected cells. All four assays demonstrated the ability to detect the presence of these functional antibody responses in a specific and reproducible manner. Interestingly, we found weak to moderate correlations between the four assays (Spearman rho ranged from 0.50 to 0.74), suggesting limited overlap in the responses captured by the individual assays. Lastly, while we initially developed each assay with multiple dilutions in an effort to capture the full relationship between antibody titers and assay outcome, we explored the relationship between fewer antibody dilutions and the full dilution series for each assay to reduce assay costs and improve assay efficiency. We found high correlations between the full dilution series and fewer or single dilutions of plasma. Use of single or fewer sample dilutions to accurately determine the response rates and magnitudes of the responses allows for high-throughput use of these assays platforms to facilitate assessment of antibody responses elicited by SARS-CoV-2 infection and vaccination in large clinical studies.

Severe acute respiratory syndrome coronavirus 2 environmental contamination in hospital rooms is uncommon using viral culture techniques.

We assessed environmental contamination of inpatient rooms housing coronavirus disease 2019 (COVID-19) patients in a dedicated COVID-19 unit. Contamination with severe acute respiratory syndrome coronavirus 2 was found on 5.5% (19/347) of surfaces via reverse transcriptase polymerase chain reaction and 0.3% (1/347) of surfaces via cell culture. Environmental contamination is uncommon in hospitals rooms; RNA presence is not a specific indicator of infectious virus.

A pan-coronavirus RT-PCR assay for rapid viral screening of animal, human, and environmental specimens.

We examined a collection of 386 animal, 451 human, and 109 archived bioaerosol samples with a new pan-species coronavirus molecular assay. Thirty-eight (4.02%) of 946 specimens yielded evidence of human or animal coronaviruses. Our findings demonstrate the utility of employing the pan-CoV RT-PCR assay in detecting varied coronavirus among human, animal, and environmental specimens. This RT-PCR assay might be employed as a screening diagnostic for early detection of coronaviruses incursions or prepandemic coronavirus emergence in animal or human populations.

TMEM41B is a host factor required for the replication of diverse coronaviruses including SARS-CoV-2.

Antiviral therapeutics are a front-line defense against virally induced diseases. Because viruses frequently mutate to escape direct inhibition of viral proteins, there is interest in targeting the host proteins that the virus must co-opt to complete its replication cycle. However, a detailed understanding of the interactions between the virus and the host cell is necessary in order to facilitate development of host-directed therapeutics. As a first step, we performed a genome-wide loss of function screen using the alphacoronavirus HCoV-229E to better define the interactions between coronaviruses and host factors. We report the identification and validation of an ER-resident host protein, TMEM41B, as an essential host factor for not only HCoV-229E but also genetically distinct coronaviruses including the pandemic betacoronavirus SARS-CoV-2. We show that the protein is required at an early, but post-receptor engagement, stage of the viral lifecycle. Further, mechanistic studies revealed that although the protein was not enriched at replication complexes, it likely contributes to viral replication complex formation via mobilization of cholesterol and other lipids to facilitate host membrane expansion and curvature. Continued study of TMEM41B and the development of approaches to prevent its function may lead to broad spectrum anti-coronavirus therapeutics.

Environmental and Aerosolized Severe Acute Respiratory Syndrome Coronavirus 2 Among Hospitalized Coronavirus Disease 2019 Patients.

During April and May 2020, we studied 20 patients hospitalized with coronavirus disease 2019 (COVID-19), their hospital rooms (fomites and aerosols), and their close contacts for molecular and culture evidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Among >400 samples, we found molecular evidence of virus in most sample types, especially the nasopharyngeal (NP), saliva, and fecal samples, but the prevalence of molecular positivity among fomites and aerosols was low. The agreement between NP swab and saliva positivity was high (89.5%; κ = 0.79). Two NP swabs collected from patients on days 1 and 7 post-symptom onset had evidence of infectious virus (2 passages over 14 days in Vero E6 cells). In summary, the low molecular prevalence and lack of viable SARS-CoV-2 virus in fomites and air samples implied low nosocomial risk of SARS-CoV-2 transmission through inanimate objects or aerosols.

A RT-PCR assay for the detection of coronaviruses from four genera.

BACKGROUND: During the past two decades, three novel coronaviruses (CoVs) have emerged to cause international human epidemics with severe morbidity. CoVs have also emerged to cause severe epidemics in animals. A better understanding of the natural hosts and genetic diversity of CoVs are needed to help mitigate these threats. OBJECTIVE: To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily. STUDY DESIGN AND RESULTS: We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 102 copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types. CONCLUSIONS: This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily.