Plaque Reduction Neutralization Test (PRNT) Accuracy in Evaluating Humoral Immune Response to SARS-CoV-2.

Massive vaccination positively impacted the SARS-CoV-2 pandemic, being a strategy to increase the titers of neutralizing antibodies (NAbs) in the population. Assessing NAb levels and understanding the kinetics of NAb responses is critical for evaluating immune protection. In this study, we optimized and validated a PRNT50 assay to assess 50% virus neutralization and evaluated its accuracy to measure NAbs to the original strain or variant of SARS-CoV-2. The optimal settings were selected, such as the cell (2 × 105 cells/well) and CMC (1.5%) concentrations and the viral input (~60 PFU/well) for PRNT-SARS-CoV-2 with cut-off point = 1.64 log5 based on the ROC curve (AUC = 0.999). The validated PRNT-SARS-CoV-2 assay presented high accuracy with an intraassay precision of 100% for testing samples with different NAb levels (low, medium, and high titers). The method displays high selectivity without cross-reactivity with dengue (DENV), measles (MV), zika (ZIKV), and yellow fever (YFV) viruses. In addition, the standardized PRNT-SARS-CoV-2 assay presented robustness when submitted to controlled variations. The validated PRNT assay was employed to test over 1000 specimens from subjects with positive or negative diagnoses for SARS-CoV-2 infection. Patients with severe COVID-19 exhibited higher levels of NAbs than those presenting mild symptoms for both the Wuhan strain and Omicron. In conclusion, this study provides a detailed description of an optimized and validated PRNT50 assay to monitor immune protection and to subsidize surveillance policies applied to epidemiologic studies of COVID-19.

Protective Immunity of COVID-19 Vaccination with ChAdOx1 nCoV-19 Following Previous SARS-CoV-2 Infection: A Humoral and Cellular Investigation.

Infections caused by SARS-CoV-2 induce a severe acute respiratory syndrome called COVID-19 and have led to more than six million deaths worldwide. Vaccination is the most effective preventative measure, and cellular and humoral immunity is crucial to developing individual protection. Here, we aim to investigate hybrid immunity against SARS-CoV-2 triggered by the ChAadOx1 nCoV-19 vaccine in a Brazilian cohort. We investigated the immune response from ChAadOx1 nCoV-19 vaccination in naïve (noCOVID-19) and previously infected individuals (COVID-19) by analyzing levels of D-dimers, total IgG, neutralizing antibodies (Nabs), IFN-γ (interferon-γ) secretion, and immunophenotyping of memory lymphocytes. No significant differences in D-dimer levels were observed 7 or 15 days after vaccination (DAV). All vaccinated individuals presented higher levels of total IgG or Nabs with a positive correlation (R = 0.88). Individuals in the COVID-19 group showed higher levels of antibody and memory B cells, with a faster antibody response starting at 7 DAV compared to noCOVID-19 at 15 DAV. Further, ChAadOx1 nCoV-19 vaccination led to enhanced IFN-γ production (15 DAV) and an increase in activated T CD4+ naïve cells in noCOVID-19 individuals in contrast with COVID-19 individuals. Hence, our data support that hybrid immunity triggered by ChAadOx1 nCoV-19 vaccination is associated with enhanced humoral response, together with a balanced cellular response.

Lactoferrin affects rhinovirus B-14 entry into H1-HeLa cells.

Lactoferrin is part of the innate immune system, with antiviral activity against numerous DNA and RNA viruses. Rhinoviruses, the leading cause of the common cold, are associated with exacerbation of respiratory illnesses such as asthma. Here, we explored the effect of bovine lactoferrin (BLf) on RV-B14 infectivity. Using different assays, we show that the effect of BLf is strongest during adhesion of the virus to the cell and entry. Tracking the internalisation of BLf and virus revealed a degree of colocalisation, although their interaction was only confirmed in vitro using empty viral particles, indicating a possible additional influence of BLf on other infection steps.

Impairing the function of MLCK, myosin Va or myosin Vb disrupts Rhinovirus B14 replication.

Together, the three human rhinovirus (RV) species are the most frequent cause of the common cold. Because of their high similarity with other viral species of the genus Enterovirus, within the large family Picornaviridae, studies on RV infectious activities often offer a less pathogenic model for more aggressive enteroviruses, e.g. poliovirus or EV71. Picornaviruses enter via receptor mediated endocytosis and replicate in the cytosol. Most of them depend on functional F-actin, Rab proteins, and probably motor proteins. To assess the latter, we evaluated the role of myosin light chain kinase (MLCK) and two myosin V isoforms (Va and Vb) in RV-B14 infection. We report that ML-9, a very specific MLCK inhibitor, dramatically reduced RV-B14 entry. We also demonstrate that RV-B14 infection in cells expressing dominant-negative forms of myosin Va and Vb was impaired after virus entry. Using immunofluorescent localization and immunoprecipitation, we show that myosin Va co-localized with RV-B14 exclusively after viral entry (15 min post infection) and that myosin Vb was present in the clusters of newly synthesized RNA in infected cells. These clusters, observed at 180 min post infection, are reminiscent of replication sites. Taken together, these results identify myosin light chain kinase, myosin Va and myosin Vb as new players in RV-B14 infection that participate directly or indirectly in different stages of the viral cycle.