ABSTRACT
Background: People with cystic fibrosis (PwCF) have chronic, pronounced respiratory damage and have been considered among those at highest risk for serious harm from SARS-CoV-2. Numerous clinical studies have reported that individuals with CF in North America and Europe, although highly susceptible to COVID-19, do not have mortality levels that exceed those of the general population. Method(s): To understand features that might influence lethality of COVID- 19 in PwCF, we tested potential relationships between CFTR and viral pathogenesis. As one approach to evaluate impact of CF transmembrane conductance regulator (CFTR) on COVID-19 severity, independent sets of blood samples fromvirally infected individualswere genotyped. Bloodwas obtained from 424 U.S. patients hospitalized with severe COVID-19 and a much larger European cohort of 7147 healthy individuals and 2587 individuals with severe COVID-19. Deoxyribonucleic acid in both studies was probed for the F508del variant. In other experiments, we investigated the possibility that lack of CFTR might alter viral binding and propagation. We used human bronchial epithelial cell (HBEC) monolayers from individuals without functional CFTR for this purpose. Finally, we examined effects of CF airway secretions and features such as viscosity, pH, and protease/anti-protease imbalance during SARS-CoV-2 infection. Result(s): We found no evidence of a relationship between deficient CFTR function (based on carrier status for the severe F508del defect) and clinical outcomes from COVID-19. In addition, viral propagation studies using airway epithelial monolayers (a model that reproduces many aspects of in vivo tissue biology) were not influenced by homozygous absence of CFTR. We show that levels of angiotensin converting enzyme-2 receptor messenger ribonucleic acid (mRNA) appear normal in CF primary epithelium, whereas transmembrane serine protease 2 mRNA is variable but lower ( p < 0.001) in a manner that correlates with viral infectivity (R2 = 0.76). Dependence of viral proliferation on features of CF mucosal fluid-including pH (viral replication optimum at pH 7-7.5), viscosity (diminished propagation in highly viscous apical media), and protease/ anti-protease imbalancewere identified as likely contributors to efficiency of SARS-CoV-2 replication and pathogenesis. Conclusion(s): These findings using patient data, CF and non-CF primary airway epithelia, and CF airway secretions fail to demonstrate a causal relationship between loss of CFTR and susceptibility to severe COVID-19. Notwithstanding the caveat that addition of virus in small buffer volumes disrupts airway surface liquid depth and composition, our findings also argue against a role for CFTR during acute infection of airway cells in vitro. On the other hand, chronic disruption of periciliary liquid, diminished pH, altered protease/anti-protease homeostasis, and increased fluid viscosity (sequelae that occur in CF lungs) were implicated as contributors to impaired SARS-CoV-2 propagation. Such studies provide a basis for future work to test relationships between CFTR and severity of COVID-19. Copyright © 2022, European Cystic Fibrosis Society. All rights reserved
ABSTRACT
While a variety of therapeutic options continue to emerge for COVID-19 treatment, convalescent plasma (CP) has been used as a possible treatment option early in the pandemic. One of the most significant challenges with CP therapy, however, both when defining its efficacy and implementing its approach clinically, is accurately and efficiently characterizing an otherwise heterogenous therapeutic treatment. Given current limitations, our goal is to leverage a SARS antibody testing platform with a newly developed automated endpoint titer analysis program to rapidly define SARS-CoV-2 antibody levels in CP donors and hospitalized patients. A newly developed antibody detection platform was used to perform a serial dilution enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig)G, IgM, and IgA SARS-CoV-2 antibodies. Data were then analyzed using commercially available software, GraphPad Prism, or a newly developed program developed in Python called TiterScape, to analyze endpoint titers. Endpoint titer calculations and analysis times were then compared between the two analysis approaches. Serial dilution analysis of SARS-CoV-2 antibody levels revealed a high level of heterogeneity between individuals. Commercial platform analysis required significant time for manual data input and extrapolated endpoint titer values when the last serial dilution was above the endpoint cutoff, occasionally producing erroneously high results. By contrast, TiterScape processed 1008 samples for endpoint titer results in roughly 14 minutes compared with the 8 hours required for the commercial software program analysis. Equally important, results generated by TiterScape and Prism were highly similar, with differences averaging 1.26 ± 0.2 percent (mean ± SD). The pandemic has created unprecedented challenges when seeking to accurately test large numbers of individuals for SARS-CoV-2 antibody levels with a rapid turnaround time. ELISA platforms capable of serial dilution analysis coupled with a highly flexible software interface may provide a useful tool when seeking to define endpoint titers in a high-throughput manner. Immunohematology 2021;37:33-43.While a variety of therapeutic options continue to emerge for COVID-19 treatment, convalescent plasma (CP) has been used as a possible treatment option early in the pandemic. One of the most significant challenges with CP therapy, however, both when defining its efficacy and implementing its approach clinically, is accurately and efficiently characterizing an otherwise heterogenous therapeutic treatment. Given current limitations, our goal is to leverage a SARS antibody testing platform with a newly developed automated endpoint titer analysis program to rapidly define SARS-CoV-2 antibody levels in CP donors and hospitalized patients. A newly developed antibody detection platform was used to perform a serial dilution enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig)G, IgM, and IgA SARS-CoV-2 antibodies. Data were then analyzed using commercially available software, GraphPad Prism, or a newly developed program developed in Python called TiterScape, to analyze endpoint titers. Endpoint titer calculations and analysis times were then compared between the two analysis approaches. Serial dilution analysis of SARS-CoV-2 antibody levels revealed a high level of heterogeneity between individuals. Commercial platform analysis required significant time for manual data input and extrapolated endpoint titer values when the last serial dilution was above the endpoint cutoff, occasionally producing erroneously high results. By contrast, TiterScape processed 1008 samples for endpoint titer results in roughly 14 minutes compared with the 8 hours required for the commercial software program analysis. Equally important, results generated by TiterScape and Prism were highly similar, with differences averaging 1.26 ± 0.2 percent (mean ± SD). The pandemic has created unprecedented challenges when seeking to accurately test large numbers of individuals for SARS-CoV-2 antibody levels with a rapid turnaround time. ELISA platforms capable of serial dilution analysis coupled with a highly flexible software interface may provide a useful tool when seeking to define endpoint titers in a high-throughput manner. Immunohematology 2021;37:3343.