Competitive multiplex binding assay can monitor SARS-CoV-2 immune responses using nanobodies

By Angela Betsaida B. Laguipo, BSNMay 3 2021

Measuring the presence and performance of neutralizing antibodies is crucial in the fight against the coronavirus disease 2019 (COVID-19) pandemic. This allows us to glimpse how effective vaccines and antibody therapies are in conferring protective immunity against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Researchers in Germany and the U.S. described 11 unique nanobodies (Nbs) specific for the SARS-CoV-2 spike receptor-binding domain (RBD), of which 8 Nbs potentially inhibit the connection of RBD with the angiotensin-converting enzyme 2 (ACE2).

The study, published in the journal Embo Reports, shows the generation of biparatopic nanobodies as surrogates to monitor neutralizing immune responses in SARS-CoV-2 infected people. A competitive multiplex binding assay, called NeutrobidyPlex, enables high-throughput screening and extensive analyses of infected or vaccinated individuals.

Study: NeutrobodyPlex—monitoring SARS‐CoV‐2 neutralizing immune responses using nanobodies. Image Credit: Huen Structure Bio / Shutterstock

The SARS-CoV-2 infectious agent

The SARS-CoV-2 contains a receptor-binding domain (RBD) located within the S1 subunit. It interacts with the angiotensin-converting enzyme (ACE2) receptor expressed on human epithelial cells of the respiratory tract to promote infection.

The spike protein and the ACE2 receptor act like a key and lock that binds to pave the way for viral entry. In-depth analysis showed the presence of spike-specific neutralizing antibodies (Nabs) in convalescent individuals who have recovered from COVID-19. These nanobodies inhibited viral uptake by several mechanisms.

To date, many nanobodies are in preclinical or clinical development to be tested if they can be prophylactic or therapeutic agents for COVID-19.

Nanobodies are single-domain antibodies derived from alpaca heavy-chain only antibodies. They have unique properties, like having a nanoscale size but a potent structure.

Compared to traditional monoclonal or polyclonal antibodies, nanobodies are smaller, more stable and consistent, have a higher affinity, and are easier to use.

Serological assays

Since the start of the pandemic, various serological SARS-CoV-2 assays have monitored seroconversion in people and estimated how many of the population have been exposed to the virus.

Most available serological tests measure the complete immune response, distinguishing between total binding and neutralizing antibodies. Detecting neutralizing antibodies is typically performed by traditional virus neutralization tests (VNTs), which are time-consuming and need work with infections virions in a specialized biosafety level 3 (BSL3) facility.

The study

To help overcome these problems, the researchers planned to use nanobodies as antibody surrogates and created a competitive binding approach to detect for neutralizing antibodies on a high‐throughput basis in samples from patients or vaccinated individuals.

The team selected 11 unique nanobodies derived from an alpaca vaccinated with glycosylated SARS‐CoV‐2 RBD. Of these, the team identified eight nanobodies that efficiently blocked that interaction between RBD, S1, and homotrimeric spike protein (S), with ACE2 and neutralized SARS-CoV-2 infection in human cells. To arrive at this finding, the team used a multiplex in vitro binding assay.

From there, the team chose two of the most robust nanobodies that target different epitopes within RBD and produced a biparatopic Nb (bipNb). The bipNb represents a strong antibody surrogate with IC50 and improved binding affinities.

The team used the bipNb in a competitive multiplex binding assay, called NeutrobodyPlex, which provided a flexible high-throughput approach to detect a neutralizing immune response in people who have been exposed to the virus or had been vaccinated.

This way, researchers could monitor the immunity status in the general population to determine if vaccine efforts were successful in curbing the pandemic.

The study findings revealed that nanobodies specific for SARS-CoV-2 Spike RBD, which inhibits its interaction and binding with ACE2, are produced. Further findings also showed that the nanobodies binding inside and outside the RBD-ACE2 interface form biparatopic nanobodies for serological SARS-CoV-2 antibody testing.

The method used, NeutrobodyPlex, showed accurate detection of neutralizing antibodies in samples. Further, it allowed for the detailed classification of the neutralization potency of patients.

“We demonstrate that NeutrobodyPlex enables high‐throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor the immune status or to guide vaccine design,” the researchers concluded in the study.

The use of the assay can help determine the extent of people who had been exposed to the virus or had been vaccinated. It can provide a bigger picture of vaccine success, showing how many people developed nanobodies against SARS-CoV-2.