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SARS-CoV-2, the causal agent of COVID-19, is a new coronavirus that has rapidly spread worldwide and significantly impacted human health by causing a severe acute respiratory syndrome boosted by a pulmonary hyperinflammatory response. Previous data from our lab showed that the newly excysted juveniles of the helminth parasite Fasciola hepatica (FhNEJ) modulate molecular routes within host cells related to vesicle-mediated transport and components of the innate immune response, which could potentially be relevant during viral infections. Therefore, the aim of the present study was to determine whether FhNEJ-derived molecules influence SARS-CoV-2 infection efficiency in Vero cells. Pre-treatment of Vero cells with a tegument-enriched antigenic extract of FhNEJ (FhNEJ-TEG) significantly reduced infection by both vesicular stomatitis virus particles pseudotyped with the SARS-CoV-2 Spike protein (VSV-S2) and live SARS-CoV-2. Pre-treatment of the virus itself with FhNEJ-TEG prior to infection also resulted in reduced infection efficiency similar to that obtained by remdesivir pre-treatment. Remarkably, treatment of Vero cells with FhNEJ-TEG after VSV-S2 entry also resulted in reduced infection efficiency, suggesting that FhNEJ-TEG may also affect post-entry steps of the VSV replication cycle. Altogether, our results could potentially encourage the production of FhNEJ-derived molecules in a safe, synthetic format for their application as therapeutic agents against SARS-CoV-2 and other related respiratory viruses.

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness., Competing Interests: NO authors have competing interests.

Background: COVID-19 convalescent plasma (CCP) is an experimental treatment against SARS-CoV-2. Although there has so far been no evidence of transmission through transfusion, pathogen reduction technologies (PRT) have been applied to CCP to mitigate risk of infectious disease. This study aims to assess the impact of methylene blue (MB) plus visible light PRT on the virus-neutralising activity of the specific antibodies against SARS-CoV-2., Material and Methods: Thirty-five plasma doses collected by plasmapheresis from COVID-19 convalescent donors were subjected to MB plus visible light PRT. Anti-SARS-CoV-2 RBD S1 epitope IgGs antibodies were quantified by ELISA. Titres of SARS-CoV-2 neutralising antibodies (NtAbs) were measured before and after the PRT process. A Spearman's correlation was run to determine the relationship between antibody neutralisation ability and SARS-CoV-2 IgG ELISA ratio. Pre- and post-inactivation neutralising antibody titres were evaluated using a Wilcoxon test., Results: The plasma pathogen reduction procedure did not diminish NtAbS titres and so did not cause a change in the viral neutralisation capacity of CCP. There was a strong correlation between pre-and post-PRT NtAbs and anti-SARS-CoV-2 IgGs titres., Discussion: Our results showed PRT with MB did not impair the CCP passive immunity preserving its potential therapeutic potency. Therefore, PRT of CCP should be recommended to mitigate the risk for transmission of transfusion-associated infectious disease. There is a good correlation between SARS-CoV-2 IgG titres determined by ELISA and the neutralising capacity. This allows blood centres to select CCP donors based on IgG ELISA titres avoiding the much more labour-intensive laboratory processes for determining neutralising antibodies.

We have detected two mutations in the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at amino acid positions 1163 and 1167 that appeared independently in multiple transmission clusters and different genetic backgrounds. Furthermore, both mutations appeared together in a cluster of 1,627 sequences belonging to clade 20E. This cluster is characterized by 12 additional single nucleotide polymorphisms but no deletions. The available structural information on the S protein in the pre- and postfusion conformations predicts that both mutations confer rigidity, which could potentially decrease viral fitness. Accordingly, we observed reduced infectivity of this spike genotype relative to the ancestral 20E sequence in vitro , and the levels of viral RNA in nasopharyngeal swabs were not significantly higher. Furthermore, the mutations did not impact thermal stability or antibody neutralization by sera from vaccinated individuals but moderately reduce neutralization by convalescent-phase sera from the early stages of the pandemic. Despite multiple successful appearances of the two spike mutations during the first year of SARS-CoV-2 evolution, the genotype with both mutations was displaced upon the expansion of the 20I (Alpha) variant. The midterm fate of the genotype investigated was consistent with the lack of advantage observed in the clinical and experimental data. IMPORTANCE We observed repeated, independent emergence of mutations in the SARS-CoV-2 spike involving amino acids 1163 and 1167, within the HR2 functional motif. Conclusions derived from evolutionary and genomic diversity analysis suggest that the co-occurrence of both mutations might pose an advantage for the virus and therefore a threat to effective control of the epidemic. However, biological characterization, including in vitro experiments and analysis of clinical data, indicated no clear benefit in terms of stability or infectivity. In agreement with this, continuous epidemiological surveillance conducted months after the first observations revealed that both mutations did not successfully outcompete other variants and stopped circulating 9 months after their initial detection. Additionally, we evaluated the potential of both mutations to escape neutralizing antibodies, finding that the presence of these two mutations on their own is not likely to confer antibody escape. Our results provide an example of how newly emerged spike mutations can be assessed to better understand the risk posed by new variants and indicate that some spike mutations confer no clear advantage to the virus despite independently emerging multiple times and are eventually displaced by fitter variants.

Cellular and humoral response to acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is on focus of research. We evaluate herein the feasibility of expanding virus-specific T cells (VST) against SARS-CoV-2 ex vivo through a standard protocol proven effective for other viruses. The experiment was performed in three different donors' scenarios: (a) SARS-CoV-2 asymptomatic infection/negative serology, (b) SARS-CoV-2 symptomatic infection/positive serology, and (c) no history of SARS-CoV-2 infection/negative serology. We were able to obtain an expanded VST product from donors 1 and 2 (1.6x and 1.8x increase of baseline VST count, respectively) consisting in CD3 + cells (80.3% and 62.7%, respectively) with CD4 + dominance (60% in both donors). Higher numbers of VST were obtained from the donor 2 as compared to donor 1. T-cell clonality test showed oligoclonal reproducible peaks on a polyclonal background for both donors. In contrast, VST could be neither expanded nor primed in a donor without evidence of prior infection. This proof-of-concept study supports the feasibility of expanding ex vivo SARS-CoV-2-specific VST from blood of convalescent donors. The results raise the question of whether the selection of seropositive donors may be a strategy to obtain cell lines enriched in their SARS-CoV-2-specificity for future adoptive transfer to immunosuppressed patients., (© 2021 Wiley Periodicals LLC.)

Assessment of commercial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoassays for their capacity to provide reliable information on sera neutralizing activity is an emerging need. We evaluated the performance of two commercially available lateral flow immunochromatographic assays (LFIC; Wondfo SARS-CoV-2 Antibody test and the INNOVITA 2019-nCoV Ab test) in comparison with a SARS-CoV-2 neutralization pseudotyped assay for coronavirus disease 2019 (COVID-19) diagnosis in hospitalized patients and investigate whether the intensity of the test band in LFIC associates with neutralizing antibody (NtAb) titers. Ninety sera were included from 51 patients with moderate to severe COVID-19. A green fluorescent protein (GFP) reporter-based pseudotyped neutralization assay (vesicular stomatitis virus coated with SARS-CoV-2 spike protein) was used. Test line intensity was scored using a 4-level scale (0 to 3+). The overall sensitivity of LFIC assays was 91.1% for the Wondfo SARS-CoV-2 Antibody test, 72.2% for the INNOVITA 2019-nCoV IgG, 85.6% for the INNOVITA 2019-nCoV IgM, and 92.2% for the NtAb assay. Sensitivity increased for all assays in sera collected beyond day 14 after symptoms onset (93.9%, 79.6%, 93.9%, and 93.9%, respectively). Reactivities equal to or more intense than the positive control line (≥2+) in the Wondfo assay had a negative predictive value of 100% and a positive predictive value of 96.4% for high NtAb 50 titers (≥1/160). Our findings support the use of LFIC assays evaluated herein, particularly the Wondfo test, for COVID-19 diagnosis. We also find evidence that these rapid immunoassays can be used to predict high SARS-CoV-2-S NtAb 50 titers., (© 2020 Wiley Periodicals LLC.)

Whether antibody levels measured by commercially available enzyme or chemiluminescent immunoassays targeting the SARS-CoV-2 spike (S) protein can act as a proxy for serum neutralizing activity remains to be established for many of these assays. We evaluated the degree of correlation between neutralizing antibodies (NtAb) binding the SARS-CoV-2 spike (S) protein and SARS-CoV-2-S-IgG levels measured by four commercial immunoassays in sera drawn from hospitalized COVID-19 patients. Ninety sera from 51 hospitalized COVID-19 patients were tested by a pseudotyped virus neutralization assay, the LIAISON SARS-CoV-2 S1/S2 IgG, the Euroimmun SARS-CoV-2 IgG ELISA, the MAGLUMI 2019-nCoV IgG, and the COVID-19 ELISA IgG assays. Overall, the results obtained with the COVID-19 ELISA IgG test showed the highest agreement with the NtAb assay (κ, 0.85; 95% CI, 0.63-1). The most sensitive tests were the pseudotyped virus NtAb assay and the COVID-19 ELISA IgG assay (92.2% for both). Overall, the degree correlation between antibody titers resulting in 50% virus neutralization (NtAb 50 ) in the pseudotyped virus assay and SARS-CoV-2 IgG levels was strong for the Euroimmun SARS-CoV-2 IgG ELISA (rho = 0.73) and moderate for the remaining assays (rho = 0.48 to 0.59). The kinetic profile of serum NtAb 50 titers could not be reliably predicted by any of the SARS-CoV-2 IgG immunoassays. The suitability of SARS-CoV-2-S-IgG commercial immunoassays for inferring neutralizing activity of sera from hospitalized COVID-19 patients varies widely across tests and is influenced by the time of sera collection after the onset of symptoms.

Background: The involvement of SARS-CoV-2 antibodies in mediating immunopathogenetic events in COVID-19 patients has been suggested. By using several experimental approaches, we investigated the potential association between SARS-CoV-2 IgGs recognizing the spike (S) protein receptor-binding domain (RBD), neutralizing antibodies (NtAb) targeting S, and COVID-19 severity., Patients and Methods: This unicenter, retrospective, observational study included 51 hospitalized patients (24 at the intensive care unit; ICU). A total of 93 sera from these patients collected at different time points from the onset of symptoms were analyzed. SARS-CoV-2 RBD IgGs were quantitated by ELISA and NtAb50 titers were measured in a GFP reporterbased pseudotyped virus platform. Demographic and clinical data, complete blood counts, as well as serum levels of ferritin, Dimer-D, C reactive protein (CRP), lactose dehydrogenase (LDH), and interleukin-6 (IL-6) were retrieved from clinical charts., Results: The overall correlation between levels of both antibody measurements was good (Rho = 0.82; P = 0 < 0.001). SARS-CoV-2 RBD IgG and NtAb50 levels in sera collected up to day 30 after the onset of symptoms were comparable between ICU and non-ICU patients (P=>0.1). Four ICU patients died; two of these achieved NtAb50 titers ≥1/160 while the other two exhibited a 1/80 titer. Very weak (Rho=>0.0-<0.2) or weak (Rho=>0.2-<0.4) correlations were observed between anti-RBD IgGs, NtAb50, and serum levels pro-inflammatory biomarkers., Conclusions: The data presented herein do not support an association between SARS-CoV-2 RBD IgG or NtAb50 levels and COVID-19 severity., (Copyright © 2020 Elsevier B.V. All rights reserved.)