Pan-Sarbecovirus Nanobodies May Improve Development of Superimmunity, Study Finds

With the emergence of the severe acute respiratory coronavirus 2 (SARS-CoV-2) and its mutated variants of concern, scientists plan to completely prevent the spread of respiratory viruses. A new study published in the bioRxiv* The preprint server found that the application of pan-sarbecovirus nanobodies shows a strong affinity towards all sarbecovirus clades and neutralized SARS-CoV-2 receptor binding domains. The result is “superimmunity” against sarbecoviruses, including the Omicron variant.

Study: Superimmunity by largely protective nanobodies against sarbecoviruses. Image Credit: Juan Gaertner / Shutterstock

The researchers note that preventing future viral outbreaks will require proactive methods that use a combination of protective and cost-effective technologies. They suggest that the small size and multiple routes of administration in nanobodies are an attractive addition to vaccines, small molecule drugs, and monoclonal antibody treatments.

The study

One llama received a recombinant vaccine containing the SARS-CoV-2 receptor binding Fc-domain fusion protein. Blood samples were taken two months after the initial dose and three booster injections. The Lama then received four more boosters in two months before collecting another round of blood samples.

Blood samples after booster immunization showed antibodies with a high affinity to the SARS-CoV-2 receptor binding domain compared to the initial immunization blood samples. In addition, a strong neutralizing power was observed against the original strain of SARS-CoV-2 as well as the Alpha and Lambda variants.

The booster injections increased the neutralizing power of beta, delta and severe acute respiratory coronavirus (SARS-CoV). To the researchers’ surprise, the vaccine boosters showed increasing binding affinity to receptor binding domains and broad neutralizing power against a sarbecovirus spectrum.

The researchers then investigated the use of broad-spectrum pan-sarbecovirus nanobodies to help inhibit sarbecoviruses. They found that 100 nanobodies exhibited a strong affinity for the receptor binding domain of SARS-CoV-2, which also exhibited cross-reactivity with other sarbecoviruses. For example, 42% of the nanobodies were bound to four different sarbecoviruses and neutralized SARS-CoV-2 at a low dose of less than 500 nanomolar.

Nanobodies are composed of several clusters and have a wide range of physicochemical properties, including isoelectric point and hydropathy. The three largest groups of nanobodies had neutralizers linked to at least three sarbecoviruses.

The binding affinity of seventeen nanobodies was tested against the receptor binding domain of five variants of SARS-CoV-2 – including Omicron – and 18 other sarbecoviruses. All of the nanobodies showed a strong binding to the variants. Sixteen of the 17 nanobodies linked to four sarbecoviruses and seven showed broad activity against all of the sarbecoviruses studied. These nanobodies are also highly specific for sarbecoviruses and have shown no cross-reactivity with high concentrations of an extract of whole human protein.

Group A Nb 2-67 showed exceptional potency against SARS-CoV-2 and its variants.

Nanobodies have shown high durability and stability because they withstood the aerosol without compromising their activity.

Researchers found five distinct epitope classes on the SARS-CoV-2 receptor binding domain through clustering of epitopes. Nanobodies did not overlap with mutations seen in Alpha, Beta, Delta, Lambda, Gamma, and Omicron, and nanobodies covered over 85% of receptor binding domain residues.

Additionally, the nanobodies appeared to lock into a 3-up conformation. Despite different epitopes, the small nanobodies allow three copies to bind to the peak trimer simultaneously.

Throughout the study, the researchers observed an overrepresentation of a certain class of nanobodies known as class II. Those collected after the first immunization show great diversity, but the class II nanobodies collected after the booster injections converged more. These nanobodies share a central conserved hydrophobic epitope with the region stabilized by a disulfide bond.

Class IIB nanobodies constitute two large groups and have the best extent and potency against sarbecoviruses. Nanobodies form hydrophobic interactions with strong binding to retained charged residues through electrostatic interactions.

Class III nanobodies destabilize the spike protein and their structure involves the targeting of a rare non-RBS epitope with a slight overlap with Nb17. Nb17 was collected after the initial blood sample and was effective against the variants. Beyond Nb17, other class III nanobodies function through a recognition motif moving to a smaller, conserved epitope.

Class IV nanobodies function via distinct scaffold orientations and sequence-specific binding arrays. They share a highly conserved and cryptic epitope accessible only in the receptor binding domain until conformation.

Class V nanobodies target the receptor binding domain through a conserved epitope embedded in the spike protein and in a region that partially overlaps with the binding motif of class IV nanobodies.

Continuous immunization produced antibodies targeting various conserved sites on the receptor binding domain. The application of ultra-high affinity nanobodies has also shown neutralizing powers.

Pan-sarbecovirus nanobodies could neutralize the virus by sterically interfering with the binding of the receptor binding domain and the N322 glycan of human ACE2.

Compared to previous nanobodies, pan-sarbecovirus nanobodies target small, flat or convex regions.

*Important Notice

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

Sara H. Byrd