ABSTRACT
Quantitative determination of neutralizing antibodies against SARS CoV2 is of paramount importance in immunodiagnostics, vaccine efficacy testing, and immune response profiling among the vaccinated population. Cost effective, rapid, easy-to-perform assays are essential to support the vaccine development process and immunosurveillance studies. Here, we describe a bead based screening assay for S1 neutralization using recombinant fluorescent proteins of hACE2 and SARS CoV2 S1, immobilized on solid beads employing nanobodies /metal-affinity tags. Nanobody-mediated capture of SARS CoV2 Spike (S1) on agarose beads served as the trap for soluble recombinant ACE2-GFPSpark, inhibited by neutralizing antibody. The first approach demonstrates single color fluorescent imaging of ACE2 GFPspark binding to His tagged S1 Receptor Binding Domain (RBD His) immobilized beads. The second approach is dual color imaging of soluble ACE2 GFPSpark to S1 Orange Fluorescent Protein (S1 OFPSpark) beads. Both methods showed a good correlation with the gold standard pseudovirion assay and can be adapted to any fluorescent platforms for screening. Life time imaging of the ACE2 GFPSpark confirmed the interaction of ACE2 and S1 OFPSpark on beads. The self-renewable source of secreted recombinant proteins from stable cells and its direct use without necessitating purification renders the platform a cost-effective and rapid one than the popular pseudovirion assay and live virus-based assays. Any laboratory with minimum expertise can rapidly perform this bead assay for neutralizing antibody detection using stable engineered cells.
ABSTRACT
Background: Covid-19 curve can be flattened by adopting mass screening protocols with aggressive testing and isolating infected populations. The current approach largely depends on RT-PCR/rapid antigen tests that require expert personnel resulting in higher costs and reduced testing frequency. Loss of smell is reported as a major symptom of Covid-19, however, a precise olfactory testing tool to identify Covid-19 patient is still lacking. Methods: To quantitatively check for the loss of smell, we developed an odor strip, COVID-Anosmia checker, spotted with gradients of coffee and lemon grass oil. We validated its efficiency in healthy and COVID-19 positive subjects. A trial screening to identify SARS-CoV-2 infected persons was also carried out to check the sensitivity and specificity of our screening tool. Results: It was observed that COVID positive participants were hyposmic instead of being anosmic when they were subjected to smelling higher odor concentration. Our tool identified 97% of symptomatic and 94% of asymptomatic COVID-19 positive subjects after excluding most confounding factors like concurrent chronic sinusitis. Further, it was possible to reliably predict COVID-19 infection by calculating a loss of smell score with 100% specificity. We coupled this tool with a mobile application, which takes the input response from the user, and can readily categorize the user in the appropriate risk groups. Conclusion: Loss of smell can be used as a reliable marker for screening for Covid-19. Our tool can rapidly quantitate anosmia, hyposmia, parosmia, and can be used as a first-line screening tool to trace out Covid-19 infection effectively.
Subject(s)
COVID-19 , Sinusitis , Olfaction Disorders , Severe Acute Respiratory SyndromeABSTRACT
SARS-CoV-2, the causative agent of COVID-19 pandemic, is an RNA virus prone to mutations. Interaction of SARS-CoV-2 Spike (S) protein with the host cell receptor, Angiotensin-I Converting Enzyme 2 (ACE2) is pivotal for attachment and entry of the virus. Yet, natural mutations acquired on S protein during the pandemic and their impact on viral infectivity, transmission dynamics and disease pathogenesis remains poorly understood. Here, we analysed 2952 SARS-CoV-2 genomes across the globe, and identified a total of 1815 non-synonymous mutations in the S-protein that fall into 54 different types. We observed that six of these distinct mutations were located in the Receptor Binding Domain (RBD) region that directly engages host ACE2. Molecular phylogenetic analysis revealed that these RBD mutations cluster into distinct phyletic clades among global subtypes of SARS-CoV-2 implying possible emergence of novel sublineages of the strain. Structure-guided homology modelling and docking analysis predicted key molecular rearrangements in the ACE2 binding interface of RBD mutants that could result in altered virus-host interactions. We propose that our findings could be significant in understanding disease dynamics and in developing vaccines, antibodies and therapeutics for COVID-19. ImportanceCOVID-19 pandemic shows considerable variations in disease transmission and pathogenesis globally, yet reasons remain unknown. Our study identifies key S-protein mutations prevailing in SARS-CoV-2 strain that could alter viral attachment and infectivity. We propose that the interplay of these mutations could be one of the factors driving global variations in COVID-19 spread. In addition, the mutations identified in this study could be an important indicator in predicting efficacies of vaccines, antibodies and therapeutics that target SARS-CoV-2 RBD-ACE2 interface.