ABSTRACT
BackgroundCombination monoclonal antibody therapy has recently been approved for prevention and treatment of severe COVID-19 infection in the UK. Available data suggests benefit is limited to those yet to mount an effective immune response from natural infection or vaccination, but concern exists around ability to make timely assessment of immune status of community-based patients.MethodsHealthcare workers were invited to undergo paired laboratory-based and rapid point-of- care (POC) lateral flow anti-spike antibody testing. Three commercial POC tests were selected to represent currently available testing methods: a split IgM/IgG anti-spike antibody test, an anti-receptor binding domain total antibody test and an anti-spike neutralisation assay. Qualitative POC colourmetric band intensities were independently scored and correlated with quantitative IgG neutralising antibody titres (Abbott Architect SARS-CoV-2 IgG Quant II chemiluminescent microparticle immunoassay [CMIA]). CMIA titres were correlated with the World Health Organisation international reference standard for neutralising antibody. Negative controls were carried out using 2018 pre-pandemic sera and post-pandemic individuals with negative CMIA results (target population).Results190 individuals (median 40 years, IQR 29-49;76.2% female) underwent paired testing, with a further 40 pre-pandemic sera tested. Assays demonstrated high performance characteristics: split IgM/IgG assay sensitivity 96.2% (95%CI 92.4.5–98.5), specificity 100.0% (95%CI 91.8–100.0);anti-receptor binding domain total antibody assay sensitivity 100.0% (95%CI 95.5–100.0), specificity 100.0% (95%CI 69.2–100.0). The neutralising antibody assay had a specificity of 97.0% (95%CI 84.2–99.9%) and strongly correlated with neutralising antibody titre (p<0.001). Probability for matched paired results was significant (McNemar’s p<0.001) while band intensity correlated strongly with neutralising titres (p<0.0001). Positive and negative predictive values for total antibody and neutralising assays were both >99%.ConclusionsPOC assays were found to be reliable predictors of both antibody status and broadly of neutralising antibody titre. Anti-S POC assays have potential to act as suitable alternatives for rapid identification of community patient immune status at presentation.
ABSTRACT
Despite the vast increase in research activity in the coronavirus field over the past two years, researchers are still heavily reliant on non-human cells, for example Vero E6, highly heterogeneous or not fully differentiated cells, such as Calu-3, or not naturally susceptible cell lines overexpressing receptor ACE2 and other accessory factors, such as TMPRSS2. Complex cell models, such as primary cell-derived air-liquid interface epithelial models are highly representative of human tissues but are expensive and time-consuming to develop and maintain and have limited suitability for high-throughput analysis. In vitro investigations of host-pathogen interactions of viruses is highly reliant on suitable cell and tissue culture models and results are only as good as the model they have been validated in. Here, we show the use of a highly characterized human kidney cell line, Caki-1, for infection with three human coronaviruses: Betacoronaviruses severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and Middle Eastern respiratory syndrome coronavirus (MERS-CoV) and Alphacoronavirus human coronavirus 229E (hCoV-229E). Caki-1 cells show equal or superior susceptibility to all three coronaviruses when compared to other commonly used cells lines for the cultivation of the respective virus. Furthermore, we used a panel of antibodies generated against 21 SARS-CoV-2-encoded proteins to identify their location in the infected Caki-1 cells using immunocytochemistry. Most importantly, Caki-1 cells are also susceptible to two other respiratory viruses, Influenza A virus and RSV, making them an ideal model for cross-comparison of not only a broad range of coronaviruses but respiratory viruses in general.
Subject(s)
Coronavirus InfectionsABSTRACT
IntroductionSerological testing can augment delayed case identification programmes for Severe Acute Respiratory Syndrome coronoravirus-2 (SARS-CoV-2). Immunoassays employ anti-nucleocapsid (anti-NP;the majority) or potentially neutralising anti-spike (including anti-receptor binding domain;anti-RBD) antibody targets, yet correlation between assays and variability arising from disease symptomatology remains unclear. We explore these possibly differential immune responses across the disease spectrum.MethodsA multicentre prospective study was undertaken via a SARS-CoV-2 delayed case identification programme (08 May-11 July 2020). Matched samples were tested for anti-NP and anti-RBD (utilising an ‘inhouse’ double-antigen bridged assay), reactivity expressed as test/cut-off binding ratios (BR) and results compared. A multivariate linear regression model analysed age, sex, symptomatology, PCR positivity, anti-NP, and anti-RBD BRs. Participants were followed up for possible reinfection.Results902 individuals underwent matched testing;109 were SARS-CoV-2 PCR swab positive. Anti-NP, anti-RBD immunoassay agreement was 87.5% (95% CI 85.3–89.6), with BRs strongly correlated (R=0.75). PCR confirmed cases were more frequently identified by anti-RBD (sensitivity 108/109, 99.1%, 95% CI 95.0–100.0) than anti-NP (102/109, 93.6%, 95% CI 87.2–97.4). Anti-RBD identified an additional 83/325 (25.5%) cases in those seronegative for anti-NP. Presence of anti-NP (p<0.0001), fever (p=0.005), or anosmia (p=0.002) were all significantly associated with an increased anti-RBD BR. Age was associated with reduced anti-RBD BR (p=0.052). Three cases with evidence of seroconversion (anti-RBD seropositive) presented with subsequent reactive PCR results, two of which coincided with first time onset of Public Heath England SARS-CoV-2 symptoms.ConclusionsSARS-CoV-2 anti-RBD shows significant correlation with anti-NP for absolute seroconversion, and BRs. Higher BRs are seen in symptomatic individuals with significantly higher levels seen in those with fever and anosmia. The degree of discordant results (12.5%) limits the use of anti-NP as a stand-alone for delayed case finding programmes. Similarly, this discordance limits the utility of non-neutralising anti-NP assays in place of potentially neutralising anti-RBD to infer possible immunity.** this abstract presentation was awarded an Honourable Mention
ABSTRACT
Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.
Subject(s)
COVID-19ABSTRACT
The spike D614G mutation increases SARS-CoV-2 infectivity, viral load, and transmission but the molecular mechanism underlying these effects remains unclear. We report here that spike is trafficked to lysosomes and that the D614G mutation enhances the lysosomal sorting of spike and the lysosomal accumulation of spike-positive punctae in SARS-CoV-2-infected cells. Spike trafficking to lysosomes is an endocytosis-independent, V-ATPase-dependent process, and spike-containing lysosomes drive lysosome clustering but display poor lysotracker labeling and reduced uptake of endocytosed materials. These results are consistent with a lysosomal pathway of coronavirus biogenesis and raise the possibility that a common mechanism may underly the D614G mutations effects on spike protein trafficking in infected cells and the accelerated entry of SARS-CoV-2 into uninfected cells.
Subject(s)
Severe Acute Respiratory SyndromeABSTRACT
In January, Mologic, embarked on a product development pathway for COVID-19 diagnostics focusing on ELISA and rapid diagnostic tests (RDTs), with anticipated funding from Wellcome Trust and DFID. 755 clinical samples from known COVID-19 patients and hospital negative controls were tested on Mologics IgG ELISA. The reported sensitivity on 191 SGUL prospectively enrolled patients was 95% on day 7 or more post diagnosis, and 97% 10 days or more post-diagnosis. A specificity panel comprising 564 samples pre-December 2019 were tested to include most common respiratory pathogens, other types of coronavirus, and flaviviruses. Specificity in this panel was 97%. This is the first in a series of Mologic products for COVID-19, which will be deployed for COVID-19 diagnosis, contact tracing and sero-epidemiological studies to estimate disease burden and transmission with a focus on ensuring access, affordability, and availability to lowest resource settings.