Researchers evaluate antibodies that inhibit SARS-CoV-2 RBD variants with a novel assay

The causative agent of coronavirus disease 2019 (COVID-19), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in Wuhan, China, in December 2019. The virus has since spread rapidly across the globe, with the World Health Organization (WHO) declaring COVID-19 a global pandemic in March 2020. To date, SARS-CoV-2 has infected over 127.3 million lives and over 2.8 million deaths.

Diverse therapeutic options are researched, and vaccines are developed at an unprecedented pace. Concern has mounted, however, about the longevity of these interventions, given the fast mutating nature of the virus and with many SARS-CoV-2 variants of concern (including B.1.1.7, P.1 and B.1.35, 501Y.V2) having emerged towards the latter part of 2020 and early 2021. These new variants present multiple mutations at key residues in the Spike (S) glycoprotein.

Study: Simultaneous evaluation of antibodies that inhibit SARS-CoV-2 RBD variants with a novel competitive multiplex assay. Image Credit: ktsdesign / Shutterstock

The SARS-CoV-2 is coronated with S proteins, a homo-trimeric transmembrane envelope glycoprotein. It binds to the human host cell receptor ACE2 (angiotensin-converting enzyme 2), facilitating the virus's entry into the host cell.

While the receptor-binding domain (RBD) on the S protein is the main target of antibodies and vaccine development, it is also the most rapidly evolving domain in the viral structure. These changes can result in the emergence of immune escape mutations, limiting the vaccines' effectiveness and antibody therapeutics.

With the emergence of SARS-CoV-2 variants of concern, it is important to focus on the gain of function, facilitating viral infectivity, transmissibility, or neutralizing antibody escape. Due to its role in the evolving variants, scientists have focused on the surveillance of SARS-CoV-2 RBD mutations.

To assess the inhibitory response of antibodies to the RBD natural variants, simultaneously, researchers from Australia developed an assay. It is a rapid, high-throughput, multiplex assay, using which the researchers demonstrated two mechanisms of immune escape.

In this study, they observed that either the antibodies failed to recognize the viral mutations, or the antibodies have reduced inhibitory capacity because of an enhanced RBD-ACE2 affinity in the variants.

A competitive approach where antibodies simultaneously compete with ACE2 for binding to the RBD may therefore more accurately reflect the physiological dynamics of infection.”

In a recent study, released on the medRxiv* server, a team of researchers discussed the potential role of a new assay in bridging a major gap in SARS-CoV-2 research. This assay can throw light on 1) the selection of complementary monoclonal antibody candidates and 2) rapid identification of the immune escape to emerging RBD variants following vaccination or natural infection.

In this study, the researchers validated this rapid, high throughput multiplex assay. They also evaluated how a selection of naturally occurring single amino acid RBD variants observed during the viral surveillance impacts the potential efficacy of monoclonal antibody therapeutics and recognition by polyclonal convalescent human plasma.

This competitive neutralization multiplex assay allows antibodies to simultaneously compete with host cell receptor ACE2 for binding to an array of RBD variants coupled to magnetic beads. The researchers see this as an approach that better captures the physiological dynamics of the interaction.

Notably, in the escape from monoclonal antibody recognition, the researchers demonstrated that while the variant N501Y was recognized at relatively high affinity by most mAbs, the inhibition to N501Y was attenuated across most mAbs, possibly due to the enhanced affinity of this variant for ACE2.

Many studies have demonstrated that post-infection, RBD-recognising potent neutralizing antibodies are elicited, that account for most plasma neutralizing activity and inhibition of the RBD-ACE2 binding. Because about 90% of this neutralizing activity occurs in the SARS-CoV-2 immune sera, these antibodies have a great potential to be clinically useful in the treatment and prevention of SARS-CoV-2 infection.

However, there is no standardized method for assessing the antibody-mediated

neutralization of SARS-CoV-2, which can account for variation in between studies. Existing bench-mark assays have challenges such as biosafety requirements, time-consuming, difficult to implement, limitations to scalability, or assaying one RDB mutant alone at one time.

Though this assay is not intended to replace gold standard SARS-CoV-2 cell-based neutralization assays, it provides a simple solution for broadly surveying the diversity of SARS-CoV-2 neutralizing specificities to an array of RBD mutants in a rapid high-throughput format, which can easily adapt to include new RBD variants as they emerge.”

Importantly, in these assays, it is not considered that antibodies physiologically may have to compete with ACE2 for binding to the RBD. Some of these RBD variants have a significantly enhanced affinity for ACE2.

This study addressed these issues and provided an assay for SARS-CoV-2 variant surveillance: a novel high-throughput RBD-ACE2 multiplex inhibition assay that measures SARS-CoV-2 NAbs to multiple RBD natural variants simultaneously.

In conclusion, the researchers revealed how this multiplex assay can serve to flag RBD variants that may have an enhanced affinity for ACE2, describe and compare the binding kinetics of several of these enhanced affinity-RBD variants, and demonstrate how this enhanced affinity to ACE2 can reduce the inhibitory capacity of both mAbs and convalescent plasma.

*Important Notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Ramya Dwivedi

Ramya has a Ph.D. in Biotechnology from the National Chemical Laboratories (CSIR-NCL), in Pune. Her work consisted of functionalizing nanoparticles with different molecules of biological interest, studying the reaction system and establishing useful applications.