Detection of Antinuclear Antibodies Targeting Intracellular Signal Transduction, Metabolism, Apoptotic Processes and Cell Death in Critical COVID-19 Patients.

Background and Objectives: The heterogeneity of the coronavirus disease of 2019 (COVID-19) lies within its diverse symptoms and severity, ranging from mild to lethal. Acute respiratory distress syndrome (ARDS) is a leading cause of mortality in COVID-19 patients, characterized by a hyper cytokine storm. Autoimmunity is proposed to occur as a result of COVID-19, given the high similarity of the immune responses observed in COVID-19 and autoimmune diseases. Here, we investigate the level of autoimmune antibodies in COVID-19 patients with different severities. Results: Initial screening for antinuclear antibodies (ANA) IgG using ELISA revealed that 1.58% (2/126) and 4% (5/126) of intensive care unit (ICU) COVID-19 cases expressed strong and moderate ANA levels, respectively. An additional sample was positive with immunofluorescence assays (IFA) screening. However, all the non-ICU cases (n=273) were ANA negative using both assays. Samples positive for ANA were further confirmed with large-scale autoantibody screening by phage immunoprecipitation-sequencing (PhIP-Seq). The majority of the ANA-positive samples showed "speckled" ANA pattern by microscopy and revealed autoantibody specificities that targeted proteins involved in intracellular signal transduction, metabolism, apoptotic processes, and cell death by PhIP-Seq; further denoting reactivity to nuclear and cytoplasmic antigens. Conclusion: Our results further support the notion of routine screening for autoimmune responses in COVID-19 patients, which might help improve disease prognosis and patient management. Further, results provide compelling evidence that ANA-positive individuals should be excluded from being donors for convalescent plasma therapy in the context of COVID-19.

Evaluation of commercially available fully automated and ELISA-based assays for detecting anti-SARS-CoV-2 neutralizing antibodies.

Rapid and accurate measurement of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-specific neutralizing antibodies (nAbs) is paramount for monitoring immunity in infected and vaccinated subjects. The current gold standard relies on pseudovirus neutralization tests which require sophisticated skills and facilities. Alternatively, recent competitive immunoassays measuring anti-SARS-CoV-2 nAbs are proposed as a quick and commercially available surrogate virus neutralization test (sVNT). Here, we report the performance evaluation of three sVNTs, including two ELISA-based assays and an automated bead-based immunoassay for detecting nAbs against SARS-CoV-2. The performance of three sVNTs, including GenScript cPass, Dynamiker, and Mindray NTAb was assessed in samples collected from SARS-CoV-2 infected patients (n = 160), COVID-19 vaccinated individuals (n = 163), and pre-pandemic controls (n = 70). Samples were collected from infected patients and vaccinated individuals 2-24 weeks after symptoms onset or second dose administration. Correlation analysis with pseudovirus neutralization test (pVNT) and immunoassays detecting anti-SARS-CoV-2 binding antibodies was performed. Receiver operating characteristic (ROC) curve analysis was generated to assess the optimal threshold for detecting nAbs by each assay. All three sVNTs showed an excellent performance in terms of specificity (100%) and sensitivity (100%, 97.0%, and 97.1% for GenScript, Dynamiker, and Mindray, respectively) in samples collected from vaccinated subjects. GenScript demonstrated the strongest correlation with pVNT (r = 0.743, R2 = 0.552), followed by Mindray (r = 0.718, R2 = 0.515) and Dynamiker (r = 0.608, R2 = 0.369). Correlation with anti-SARS-CoV-2 binding antibodies was variable, but the strongest correlations were observed between anti-RBD IgG antibodies and Mindray (r = 0.952, R2 = 0.907). ROC curve analyses demonstrated excellent performance for all three sVNT assays in both groups, with an AUC ranging between 0.99 and 1.0 (p < 0.0001). Also, it was shown that the manufacturer's recommended cutoff values could be modified based on the tested cohort without significantly affecting the sVNT performance. The sVNT provides a rapid, low-cost, and scalable alternative to conventional neutralization assays for measuring and expanding nAbs testing across various research and clinical settings. Also, it could aid in evaluating actual protective immunity at the population level and assessing vaccine effectiveness to lay a foundation for boosters' requirements.

Comparative analysis of within-host diversity among vaccinated COVID-19 patients infected with different SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a rapidly evolving RNA virus that mutates within hosts and exists as viral quasispecies. Here, we evaluated the within-host diversity among vaccinated and unvaccinated individuals (n = 379) infected with different SARS-CoV-2 Variants of Concern. The majority of samples harbored less than 14 intra-host single-nucleotide variants (iSNVs). A deep analysis revealed a significantly higher intra-host diversity in Omicron samples than in other variants (p value < 0.05). Vaccination status and type had a limited impact on intra-host diversity except for Beta-B.1.315 and Delta-B.1.617.2 vaccinees, who exhibited higher diversity than unvaccinated individuals (p values: <0.0001 and <0.0021, respectively). Three immune-escape mutations were identified: S255F in Delta and R346K and T376A in Omicron-B.1.1.529. The latter 2 mutations were fixed in BA.1 and BA.2 genomes, respectively. Overall, the relatively higher intra-host diversity among vaccinated individuals and the detection of immune-escape mutations, despite being rare, suggest a potential vaccine-induced immune pressure in vaccinated individuals.

Validation of a Novel Fluorescent Lateral Flow Assay for Rapid Qualitative and Quantitative Assessment of Total Anti-SARS-CoV-2 S-RBD Binding Antibody Units (BAU) from Plasma or Fingerstick Whole-Blood of COVID-19 Vaccinees.

Background: Limited commercial LFA assays are available to provide a reliable quantitative measurement of the total binding antibody units (BAU/mL) against the receptor-binding domain of the SARS-CoV-2 spike protein (S-RBD). Aim: This study aimed to evaluate the performance of the fluorescence LFA FinecareTM 2019-nCoV S-RBD test along with its reader (Model No.: FS-113) against the following reference methods: (i) the FDA-approved GenScript surrogate virus-neutralizing assay (sVNT); and (ii) three highly performing automated immunoassays: BioMérieux VIDAS®3, Ortho VITROS®, and Mindray CL-900i®. Methods: Plasma from 488 vaccinees was tested by all aforementioned assays. Fingerstick whole-blood samples from 156 vaccinees were also tested by FinecareTM. Results and conclusions: FinecareTM showed 100% specificity, as none of the pre-pandemic samples tested positive. Equivalent FinecareTM results were observed among the samples taken from fingerstick or plasma (Pearson correlation r = 0.9, p < 0.0001), suggesting that fingerstick samples are sufficient to quantitate the S-RBD BAU/mL. A moderate correlation was observed between FinecareTM and sVNT (r = 0.5, p < 0.0001), indicating that FinecareTM can be used for rapid prediction of the neutralizing antibody (nAb) post-vaccination. FinecareTM BAU results showed strong correlation with VIDAS®3 (r = 0.6, p < 0.0001) and moderate correlation with VITROS® (r = 0.5, p < 0.0001) and CL-900i® (r = 0.4, p < 0.0001), suggesting that FinecareTM can be used as a surrogate for the advanced automated assays to measure S-RBD BAU/mL.