The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the ...true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.
BACKGROUND
Delayed, large‐volume bacterial culture and amotosalen/ultraviolet‐A light pathogen reduction are effective at reducing the risk of bacterial proliferation in platelet concentrates (PCs). ...Hemovigilance programs continue to receive reports of suspected septic transfusion reactions, most with low imputability. Here, we compile national hemovigilance data to determine the relative efficacy of these interventions.
STUDY DESIGN AND METHODS
Annual reports from the United Kingdom, France, Switzerland, and Belgium were reviewed between 2005 and 2016 to assess the risk of bacterial contamination and septic reactions.
RESULTS
Approximately 1.65 million delayed, large‐volume bacterial culture‐screened PCs in the United Kingdom and 2.3 million amotosalen/ultraviolet‐A–treated PCs worldwide were issued with no reported septic fatalities. One definite, one possible, and 12 undetermined/indeterminate septic reactions and eight contaminated “near misses” were reported with delayed, large‐volume bacterial cultures between 2011 and 2016, for a lower false‐negative culture rate than that in the previous 5 years (5.4 vs. 16.3 per million: odds ratio, 3.0; 95% confidence interval, 1.4‐6.5). Together, the Belgian, Swiss, and French hemovigilance programs documented zero probable or definite/certain septic reactions with 609,290 amotosalen/ultraviolet‐A–treated PCs (<1.6 per million). The rates were significantly lower than those reported with concurrently transfused, nonpathogen‐reduced PCs in Belgium (<4.4 vs. 35.6 per million: odds ratio, 8.1; 95% confidence interval,1.1‐353.3) and with historic septic reaction rates in Switzerland (<6.0 vs. 82.9 per million: odds ratio, 13.9; 95% confidence interval, 2.1‐589.2), and the rates tended to be lower than those from concurrently transfused, nonpathogen‐reduced PCs in France (<4.7 vs. 19.0 per million: odds ratio, 4.1; 95% confidence interval, 0.7‐164.3).
CONCLUSION
Pathogen reduction and bacterial culture both reduced the incidence of septic reactions, although under‐reporting and strict imputability criteria resulted in an underestimation of risk.
A coronavirus antigen microarray (COVAM) was constructed containing 11 SARS-CoV-2, 5 SARS-1, 5 MERS, and 12 seasonal coronavirus recombinant proteins. The array is designed to measure immunoglobulin ...isotype and subtype levels in serum or plasma samples against each of the individual antigens printed on the array. We probed the COVAM with COVID-19 convalescent plasma (CCP) collected from 99 donors who recovered from a PCR+ confirmed SARS-CoV-2 infection. The results were analyzed using two computational approaches, a generalized linear model (glm) and random forest (RF) prediction model, to classify individual specimens as either Reactive or non-reactive against the SARS-CoV-2 antigens. A training set of 88 pre-COVID-19 specimens (PreCoV) collected in August 2019 and102 positive specimens from SARS-CoV-2 PCR+ confirmed COVID-19 cases was used for these analyses. Results compared with an FDA emergency use authorized (EUA) SARS-CoV2 S1-based total Ig chemiluminescence immunoassay (Ortho Clinical Diagnostics VITROS Anti-SARS-CoV-2 Total, CoV2T) and with a SARS-CoV-2 S1-S2 spike-based pseudovirus micro neutralization assay (SARS-CoV-2 reporter viral particle neutralization titration (RVPNT) showed high concordance between the three assays. Three CCP specimens that were negative by the VITROS CoV2T immunoassay were also negative by both COVAM and the RVPNT assay. Concordance between VITROS CoV2T and COVAM was 96%, VITROS CoV2T and RVPNT 93%, and RVPNT and COVAM 91%. The discordances were all weakly reactive samples near the cutoff threshold of the VITROS CoV2T immunoassay. The multiplex COVAM allows CCP to be grouped according to antibody reactivity patterns against 11 SARS-CoV-2 antigens. Unsupervised K-means analysis, via the gap statistics, as well as hierarchical clustering analysis revealed three main clusters with distinct reactivity intensities and patterns. These patterns were not recapitulated by adjusting the VITROS CoV2T or RVPNT assay thresholds. Plasma classified by COVAM reactivity patterns offers potential to improve CCP therapeutic efficacy CoV2T alone. The use of a SARS-CoV-2 antigen array can qualify CCP for administration as a treatment for acute COVID-19, and interrogate vaccine immunogenicity and performance in preclinical, clinical studies, and routine vaccination to identify antibody responses predictive of protection from infection and disease.
Abstract
An easily implementable serological assay to accurately detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing antibodies is urgently needed to better track herd ...immunity, vaccine efficacy and vaccination rates. Herein, we report the Split-Oligonucleotide Neighboring Inhibition Assay (SONIA) which uses real-time qPCR to measure the ability of neutralizing antibodies to block binding between DNA-barcoded viral spike protein subunit 1 and the human angiotensin-converting enzyme 2 receptor protein. The SONIA neutralizing antibody assay using finger-prick dried blood spots displays 91–97% sensitivity and 100% specificity in comparison to the live-virus neutralization assays using matched serum specimens for multiple SARS-CoV-2 variants-of-concern. The multiplex version of this neutralizing antibody assay, using easily collectable finger-prick dried blood spots, can be a valuable tool to help reveal the impact of age, pre-existing health conditions, waning immunity, different vaccination schemes and the emergence of new variants-of-concern.
The tragedy of transfusion-associated hepatitis and HIV spurred a decades-long overhaul of the regulatory oversight and practice of blood transfusion. Consequent to improved donor selection, testing, ...process control, clinical transfusion practice and post-transfusion surveillance, transfusion in the United States and other high-income countries is now a very safe medical procedure. Nonetheless, pathogens continue to emerge and threaten the blood supply, highlighting the need for a proactive approach to blood transfusion safety. Blood donor populations and the global transfusion infrastructure are under-utilized resources for the study of infectious diseases. Blood donors are large, demographically diverse subsets of general populations for whom cross-sectional and longitudinal samples are readily accessible for serological and molecular testing. Blood donor collection networks span diverse geographies, including in low- and middle-income countries, where agents, especially zoonotic pathogens, are able to emerge and spread, given limited tools for recognition, surveillance and control. Routine laboratory storage and transportation, coupled with data capture, afford access to rich epidemiological data to assess the epidemiology and pathogenesis of established and emerging infections. Subsequent to the State of the Science in Transfusion Medicine symposium in 2022, our working group (WG), "Emerging Infections: Impact on Blood Science, the Blood Supply, Blood Safety, and Public Health" elected to focus on "leveraging donor populations to study the epidemiology and pathogenesis of transfusion-transmitted and emerging infectious diseases." The 5 landmark studies span (1) the implication of hepatitis C virus in post-transfusion hepatitis, (2) longitudinal evaluation of plasma donors with incident infections, thus informing the development of a widely used staging system for acute HIV infection, (3) explication of the dynamics of early West Nile Virus infection, (4) the deployment of combined molecular and serological donor screening for Babesia microti, to characterize its epidemiology and infectivity and facilitate routine donor screening, and (5) national serosurveillance for SARS-CoV-2 during the COVID-19 pandemic. The studies highlight the interplay between infectious diseases and transfusion medicine, including the imperative to ensure blood transfusion safety and the broader application of blood donor populations to the study of infectious diseases.
Abstract
Background
Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2)‐infected patients exhibit disease ranging from asymptomatic to severe pneumonia, multi‐organ failure, and death. ...convalescent COVID plasma (CCP) from recovered patients with high levels of neutralizing antibodies has demonstrated therapeutic efficacy to reduce the morbidity of coronavirus disease 2019 (COVID‐19) in some studies. The development of assays to characterize the activity of CCP to neutralize SARS‐CoV‐2 infectivity offers the possibility to improve potential therapeutic efficacy. Lyophilization of CCP may increase the availability of this therapy. We hypothesized that SARS‐CoV‐2 antibody profiles of pooled lyophilized pathogen‐reduced CCP from COVID‐19‐recovered blood donors retains virus‐neutralizing efficacy as reported for frozen pathogen‐reduced CCP.
Methods
Pooled lyophilized pathogen‐reduced plasma was prepared from recovered COVID plasma donors. Antibodies to SARS‐CoV‐2 were characterized in each donor plasma prior to pathogen reduction and lyophilization and after lyophilization of individual CCP, and in the lyophilized CCP pool. Several complimentary assays were used to characterize antibody levels, neutralizing capacity, and the spectrum of antigen reactivity. The mean values for individual plasma samples and the value in the pool were compared.
Results
The mean ratio for antibody binding to SARS‐CoV‐2 antigens before and after treatment was 0.95 ± 0.22 mean fluorescent intensity (MFI) units. Antibody activity to an array of influenza virus antigens demonstrated a mean activity ratio of 0.92 ± 0.12 MFI before and after treatment.
Conclusions
The antibody activity in pooled pathogen‐reduced lyophilized CCPs demonstrated minimal impact due to pathogen reduction treatment and lyophilization.
INTRODUCTION
Transfusion‐associated graft‐versus‐host disease (TA‐GVHD) is a rare complication after transfusion of components containing viable donor T cells. Gamma irradiation with doses that stop ...T‐cell proliferation is the predominant method to prevent TA‐GVHD. Treatment with pathogen inactivation methodologies has been found to also be effective against proliferating white blood cells, including T cells. In this study, T‐cell inactivation was compared, between amotosalen/ultraviolet A (UVA) treatment and gamma‐irradiation (2500 cGy), using a sensitive limiting dilution assay (LDA) with an enhanced dynamic range.
METHODS AND MATERIALS
Matched plasma units (N = 8), contaminated with 1 × 106 peripheral blood mononuclear cells (PBMCs) per mL, were either treated with amotosalen/UVA or gamma irradiation, or retained as untreated control. Posttreatment, cells were cultured under standardized conditions. T‐cell proliferation was determined by the incorporation of 3H‐thymidine and correlated with microscopic detection.
RESULTS
Range‐finding experiments showed that after gamma irradiation (2500 cGy), significant T‐cell proliferation could be observed at a 1 × 107 cell culture density, some proliferation at 1 × 106, and none at 1 × 105 cells/well. Based on these facts, a quantitative comparison was carried out between amotosalen/UVA at the highest challenge of 1 × 107 PBMCs/well, and gamma irradiation at 1 × 106 and 1 × 105 PBMCs/well. Complete inactivation of the T cells after amotosalen/UVA treatment was observed, equivalent to greater than 6.2 log inactivation. Complete inactivation of the T cells was also observed after gamma irradiation when 1 × 105 PBMCs/well were cultured (>4.2 log inactivation). Proliferation was observed when 1 × 106 PBMCs/well were cultured (≤5.2 log inactivation) after gamma irradiation.
CONCLUSION
Amotosalen/UVA treatment more effectively inactivates T cells than the current standard of gamma irradiation (2500 cGy) for the prevention of TA‐GVHD.
BACKGROUND
In 2014, passive immunization by transfusion of Ebola convalescent plasma (ECP) was considered for treating patients with acute Ebola virus disease (EVD). Early Ebola virus (EBOV) ...seroconversion confers a survival advantage in natural infection, hence transfusion of ECP plasma with high levels of neutralizing EBOV antibodies is a potential passive immune therapy. Techniques to reduce the risk of other transfusion‐transmitted infections (TTIs) are warranted as recent ECP survivors are ineligible as routine blood donors. As part of an ongoing clinical trial to evaluate the safety and effectiveness of ECP, the impact of amotosalen/UVA pathogen reduction technology (PRT) on EBOV antibody characteristics was examined.
STUDY DESIGN AND METHODS
Serum and plasma samples were collected from EVD‐recovered subjects at multiple timepoints and evaluated by ELISA for antibodies to recombinant EBOV glycoprotein (GP) and irradiated whole EBOV antigen, as well as for EBOV microneutralization, classic plaque reduction neutralization test (PRNT) and EBOV pseudovirion neutralization assay (PsVNA) activity.
RESULTS
Six subjects donated 40 individual ECP units. Substantial antibody titers and neutralizing activity results were demonstrated but were generally lower for the ACD plasma samples compared to the serum samples. Anti‐EBOV titers by all assays remained essentially unchanged after PRT.
CONCLUSION
Treatment of ECP with PRT to reduce the risk of TTI did not significantly reduce EBOV IgG antibody titers or neutralizing activity. Although ECP was used in the treatment of repatriated patients, no PRT units from this study were transfused to EVD patients. This inventory of PRT‐treated ECP is currently available for future clinical evaluation.
Background
COVID‐19 convalescent plasma (CCP), from donors recovered from severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection, is one of the limited therapeutic options currently ...available for the treatment of critically ill patients with COVID‐19. There is growing evidence that CCP may reduce viral loads and disease severity; and reduce mortality. However, concerns about the risk of transfusion‐transmitted infections (TTI) and other complications associated with transfusion of plasma, remain. Amotosalen/UVA pathogen reduction treatment (A/UVA‐PRT) of plasma offers a mitigation of TTI risk, and when combined with pooling has the potential to increase the diversity of the polyclonal SARS‐CoV‐2 neutralizing antibodies.
Study design and methods
This study assessed the impact of A/UVA‐PRT on SARS‐CoV‐2 antibodies in 42 CCP using multiple complimentary assays including antigen binding, neutralizing, and epitope microarrays. Other mediators of CCP efficacy were also assessed.
Results
A/UVA‐PRT did not negatively impact antibodies to SARS‐CoV‐2 and other viral epitopes, had no impact on neutralizing activity or other potential mediators of CCP efficacy. Finally, immune cross‐reactivity with other coronavirus antigens was observed raising the potential for neutralizing activity against other emergent coronaviruses.
Conclusion
The findings of this study support the selection of effective CCP combined with the use of A/UVA‐PRT in the production of CCP for patients with COVID‐19.
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