ABSTRACT
Quantitative determination of neutralizing antibodies against Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) is paramount 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. 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-CoV-2-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.
ABSTRACT
Following the recent clinical clearance of an Indian DNA COVID-19 vaccine, India and Africa are potential regions where DNA vaccines may become a major delivery system subject to a range of immunological and regulatory scrutiny. The ongoing COVID pandemic highlights the need to tackle viral variants and expand the number of antigens and assess diverse delivery systems. To address some of these key issues, we have created a Dengue DNA vaccine candidate with the EDIII region as the key antigen given the promise of this segment in not causing ADE, a challenge with this disease. In addition, we have added the NS1 region to broaden the immune response. Following a large Dengue viral sequencing exercise in India, complemented with data from east Africa, our approach was to generate a consensus of four serotypes ED3-NS1 vaccine to explore tackling the issue of diversity. Our In silico structural analysis of EDIII consensus vaccine sequence revealed that epitopes are structurally conserved and immunogenic across HLA diversity. Vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Furthermore, the DNA vaccination confers protection against DENV challenge in AG129 mice. Finally, assaying of intracellular staining for IFN-γ, immunoglobulin IgG2(a/c) /IgG1 ratios as well as immune gene profiling suggested a strong Th1-dominant immune response. Our Dengue DNA platform with a focus on Indo-African sequences offers an approach for assessing cross reactive immunity in animal models and lays the foundation for human vaccine roll out either as a stand-alone or mix and match strategy.