Dataset Information

A bioluminescent and homogeneous SARS-CoV-2 spike RBD and hACE2 interaction assay for antiviral screening and monitoring patient neutralizing antibody levels.

ABSTRACT: Here we describe a homogeneous bioluminescent immunoassay based on the interaction between Fc-tagged SARS-CoV-2 Spike RBD and human ACE2, and its detection by secondary antibodies labeled with NanoLuc luciferase fragments LgBit and SmBit. The assay utility for the discovery of novel inhibitors was demonstrated with a panel of anti-RBD antibodies, ACE2-derived miniproteins and soluble ACE2. Studying the effect of RBD mutations on ACE2 binding showed that the N501Y mutation increased RBD apparent affinity toward ACE2 tenfold that resulted in escaping inhibition by some anti-RBD antibodies. In contrast, while E484K mutation did not highly change the binding affinity, it still escaped antibody inhibition likely due to changes in the epitope recognized by the antibody. Also, neutralizing antibodies (NAbs) from COVID-19 positive samples from two distinct regions (USA and Brazil) were successfully detected and the results further suggest the persistence of NAbs for at least 6 months post symptom onset. Finally, sera from vaccinated individuals were tested for NAbs and showed varying neutralizing activity after first and second doses, suggesting the assay can be used to assess immunity of vaccinated populations. Our results demonstrate the broad utility and ease of use of this methodology both for drug discovery and clinical research applications.

SUBMITTER: Alves J

PROVIDER: S-EPMC8445915 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:BackgroundThere is always a need for a safe and efficient vaccine platform, especially when facing a pandemic such as COVID-19. Most of the SARS-CoV-2-based vaccines are based on the full spike protein, which is presented as a trimerized protein, and many viral vector vaccines express the spike protein into the host cells and do not display it on virus surfaces. However, the spike receptor-binding domain (RBD)-based vaccines are efficient and are currently under investigation and clinical trials.MethodologyIn this study, we are testing the efficacy of the RBD displayed on a baculovirus as a mean to formulate a safe and stable carrier to induce the immune system against SARS-CoV-2. Therefore, two pseudotyped baculoviruses were constructed to display the RBD, AcRBD-sfGFP-64, and AcRBD-sfGFP-V, using two different displaying strategies based on gp64 and VSV-G envelope glycoproteins, from Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and vesicular stomatitis virus (VSV), respectively. BALB/C mice were immunized with the pseudotyped baculoviruses in a dose-optimized manner. Dot blot and Western blot were used to screen and validate the polyclonal antibodies' specificity to the SARS-CoV-2 RBD. A plaque reduction neutralization test (PRNT) was used to measure the sera neutralization capacity against a SARS-CoV-2 wild-type isolate from Egypt. ELISA was used to quantify certain cytokines for the assessment of the immune response.ResultThe outcome of our investigation showed that the monomeric RBD proteins were properly displayed on baculovirus and efficiently triggered the mouse immune system. The produced sera efficiently neutralized about 50% of SARS-CoV-2 in more than 100-fold serum dilution. The immunized mice showed a significant increase (p<0.01) in the levels of IL-2 and IFN-γ and a significant decrease (p<0.01) and (p<0.001) in the levels of IL-4 and IL-10, respectively, which suggest that AcRBD-sfGFP-64 and AcRBD-sfGFP-V induce Th1 cellular immune response.ConclusionThe produced recombinant viruses can induce the immune response without adjuvant, which needs dose optimization and further stability tests. Neutralizing antibodies were induced without affecting the health of immunized mice. Th1 response can be attainable through the system, which is of great benefit in SARS CoV-2 infection and the system can be tested for future applications including vaccine development and polyclonal antibody production.

Project description:Objective: Nobody knows when the COVID-19 pandemic will end or when and where the next coronavirus will outbreak. Therefore, it is still necessary to develop SARS-CoV-2 inhibitors for different variants or even the new coronavirus. Since SARS-CoV-2 uses its surface spike-protein to recognize hACE2, mediating its entry into cells, ligands that can specifically recognize the spike-protein have the potential to prevent infection. Methods: We have recently discovered DNA aptamers against the S2-domain of the WT spike-protein by exploiting the selection process called SELEX. After optimization, among all candidates, the aptamer S2A2C1 has the shortest sequence and the best binding affinity toward the S2-protein. More importantly, the S2A2C1 aptamer does not bind to the RBD of the spike-protein, but it efficiently blocks the spike-protein/hACE2 interaction, suggesting an RBD-independent inhibition approach. To further improve its performance, we conjugated the S2A2C1 aptamer with a reported anti-RBD aptamer, S1B6C3, using various linkers and constructed hetero-bivalent fusion aptamers. Binding affinities of mono and fusion aptamers against the spike-proteins were measured. The inhibition efficacies of mono and fusion aptamers to prevent the hACE2/spike-protein interaction were determined using ELISA. Results: Anti-spike-protein aptamers, including S2A2C1 and S1B6C3-A5-S2A2C1, maintained high binding affinity toward the WT, Delta, and Omicron spike-proteins and high inhibition efficacies to prevent them from binding to hACE2, rendering them well-suited as diagnostic and therapeutic molecular tools to target SARS-CoV-2 and its variants. Conclusions: Overall, we discovered the anti-S2 aptamer, S2A2C1, which inhibits the hACE2/spike-protein interaction via an RBD-independent approach. The anti-S2 and anti-RBD aptamers were conjugated to obtain the fusion aptamer, S1B6C3-A5-S2A2C1, which recognizes the spike-protein by an RBD-dependent approach. Our strategies, which discovered aptamer inhibitors targeting the highly conserved S2-protein, as well as the design of fusion aptamers, can be used to target new coronaviruses as they emerge.

Dataset Information

A bioluminescent and homogeneous SARS-CoV-2 spike RBD and hACE2 interaction assay for antiviral screening and monitoring patient neutralizing antibody levels.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets