Engineering nanobodies as rescuers when SARS-CoV-2 variants attack

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Scientists are pursuing a new strategy in the prolonged fight against the SARS-CoV-2 virus by designing nanobodies capable of neutralizing viral variants in two different ways.

In laboratory studies, researchers have identified two groups of molecules effective against viral variants. Using different mechanisms, nanobodies in each group bypassed mutations and deactivated the virus’s ability to bind to the receptor that allows it to enter host cells.

Although vaccination allows the resumption of some pre-pandemic activities in some parts of the world, SARS-CoV-2 quickly bypasses vaccines by mutating itself. In this study, nanobodies neutralized three emerging variants: Alpha, Beta and Gamma.

“Companies have already started to introduce the worrisome variants in building booster shots of existing vaccines,” said Kai Xu, assistant professor of veterinary biosciences at Ohio State University and co-lead author of the research. “But the virus is constantly changing, and the rate of mutation may be faster than what we can capture. Therefore, we have to use several mechanisms to control the spread of the virus. “

A time-lapse preview of the study article is published online in Nature.

Nanobodies are antibodies derived from the immunization of camelid mammals – such as camels, llamas, and alpacas – that can be engineered into tiny molecules that mimic the structures and functions of human antibodies.

For this work, the researchers immunized llamas to produce single chain antibodies against SARS-CoV-2. They also immunized transgenic “nanomice” mice, with a camelid gene that had been designed by researcher Jianliang Xu in the laboratory of Rafael Casellas, senior researcher at the National Institute of Arthritis and Musculoskeletal Diseases and cutaneous. (NIAMS), to generate nanobodies similar to those produced by camels.

The team improved the potency of nanobodies by first immunizing animals with the receptor binding domain (RBD), part of the viral surface spike protein, and then with booster injections containing all of the protein. peak.

“Using this sequential immunization strategy, we have generated nanobodies that can capture the virion by recognizing the receptor binding domain with very high affinity,” said Xu.

Scientists tested the neutralizing ability of different nanobodies, mapping the surface of RBD, performing functional and structural analyzes, and measuring the strength of their affinity to reduce candidate molecules from a large library to six.

The coronavirus is highly infectious because it binds very tightly to the ACE2 receptor to access lung and nasal cells in humans, where it copies itself to infect other cells. The receptor binding domain on the spike protein is fundamental to its success in binding to ACE2.

“This RBD-ACE2 interface sits at the top of the receptor binding domain – this region is the primary target for protective human antibodies, generated by vaccination or previous infection, to block viral entry,” said Xu. “But it is also a region frequently mutated in variants.”

The way the mutants have emerged so far suggests that the long-term dependence of current vaccines will eventually be compromised, the researchers say, as the effectiveness of antibodies is significantly affected by these mutants at the interface.

“We have found that some nanobodies can recognize a conserved region of the receptor binding domain, a hidden location too narrow for human antibodies to reach,” said Xu. And attaching there, even though it’s some distance from where RBD connects to ACE2, still accomplishes what is intended – preventing SARS-CoV-2 from entering a host cell.

The other group of nanobodies, attracted by the RBD-ACE2 interface, while in their original form could not neutralize some variants. However, when the researchers designed this group to be homotrimers – three copies linked in tandem – the nanobodies achieved a powerful neutralization of the virus. Altering the structure of nanobodies that attached to the conserved region of RBD in the same way also improved their efficiency.

There is much more research to come, but the results suggest that nanobodies could be promising tools for preventing COVID-19 mortality when vaccines are compromised, Xu said.

“Our future plan is to further isolate antibodies specifically against emerging variants for therapeutic development, and to find a better solution for vaccines by learning from these antibodies,” he said.

An NIH HIV vaccine researcher before joining the State of Ohio, Xu collaborated with several laboratories on this research. Jianliang Xu and Rafael Casellas of NIAMS, and Peter Kwong of the National Institute of Allergy and Infectious Diseases, are also equal contributors to the study. In addition to many NIH agencies, co-authoring institutions include Rockefeller University, the Aaron Diamond AIDS Research Center at Columbia University, and the Frederick National Laboratory for Cancer Research.

This work was supported by NIAMS, the National Cancer Institute, NIH Helix Systems, NIAID, and the Frederick National Laboratory for Cancer Research.

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