ABSTRACT
A key in controlling the SARS-CoV-2 pandemic is the assessment of the immune status of the population. We explored the utility of SARS-CoV-2 virus-like particles (VLPs) as antigens to detect specific humoral immune reactions in an enzyme-linked immunosorbent assay (ELISA). For this purpose, SARS-CoV-2 VLPs were produced from an engineered cell line and characterized by western blot, ELISA and nanoparticle tracking analysis. Subsequently, we collected 42 serum samples from before the pandemic (2014), 89 samples from healthy-, and 38 samples from vaccinated subjects. Seventeen samples were collected less than three weeks after infection, and 44 samples more than three weeks after infection. All serum samples were characterized for their reactivity with VLPs and the SARS-CoV-2 N- and S-protein. Finally, we compared the performance of the VLP-based ELISA with a certified in vitro diagnostic device (IVD). In the applied set of samples, we determined a sensitivity of 95.5 % and a specificity of 100 % for the certified IVD. There were 7 samples with an uncertain outcome. Our VLP-ELISA showed superior performance with a sensitivity of 97.5 %, a specificity of 100 %, and only 3 uncertain outcomes. This result warrants further research to develop a certified IVD based on SARS-CoV-2 VLPs as an antigen.
Subject(s)
Severe Acute Respiratory SyndromeABSTRACT
IntroductionThe ongoing COVID-19 pandemic situation caused by SARS-CoV-2 and variants of concern such as B.1.617.2 (Delta) and recently, B.1.1.529 (Omicron) is posing multiple challenges to humanity. The rapid evolution of the virus requires adaptation of diagnostic and therapeutic applications. ObjectivesIn this study, we describe camelid heavy-chain-only antibodies (hcAb) as useful for novel in vitro diagnostic assays and for therapeutic applications due to their neutralizing capacity. MethodsFive antibody candidates were selected out of a naive camelid library by phage display and expressed as full-length IgG2 antibodies. The antibodies were characterized by Western blot, enzyme-linked immunosorbent assays, surface plasmon resonance with regard to their specificity to the recombinant SARS-CoV-2 Spike protein and to SARS-CoV-2 virus-like particles. Neutralization assays were performed with authentic SARS-CoV-2 and pseudotyped viruses (wildtype and Omicron). ResultsAll antibodies efficiently detect recombinant SARS-CoV-2 Spike protein and SARS-CoV-2 virus-like particles in different ELISA setups. The best combination was shown with hcAb B10 as catcher antibody and HRP-conjugated hcAb A7.2 as the detection antibody. Further, four out of five antibodies potently neutralized authentic wildtype SARS-CoV-2 and particles pseudotyped with the SARS-CoV-2 Spike proteins of the wildtype and Omicron variant, sublineage BA.1 at concentrations between 0.1 and 0.35 ng/mL (ND50). ConclusionCollectively, we report novel camelid hcAbs suitable for diagnostics and potential therapy. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY
Subject(s)
Severe Acute Respiratory Syndrome , COVID-19ABSTRACT
Background: COVID 19 is associated with a hypercoagulable state and frequent thromboembolic complications. For how long this acquired abnormality lasts potentially requiring preventive measures, such as anticoagulation remains to be delineated.Methods: We used viscoelastic rotational thrombelastometry (ROTEM) in a single center cohort of 13 critical ill patients and performed follow up examinations three months after discharge from ICU.Results: We found clear signs of a hypercoagulable state due to severe hypofibrinolysis and a high rate of thromboembolic complications during the phase of acute illness. Three month follow up revealed a normalization of the initial coagulation abnormality together without evidence of venous thrombosis in all thirteen patients. Conclusion: In our cohort the coagulation profile was completely normalized three months after COVID-19. It thus appears reasonable that anticoagulation can be discontinued beyond this timepoint in patients with complete venous reperfusion.