Bacterial Superglue Enables Antiviral Antibody Discovery

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Winter the llama grabbed headlines recently for her part in generating a special type of anti-SARS-CoV-2 antibody. But in fact, any camelid has what it takes to create such valuable proteins, known as single-domain antibodies (sdAbs).

These tiny proteins, which only sharks and camelids (llamas, camels, and related species) are known to make, differ from the antibodies found in humans and other animals in that they’re encoded by just one gene instead of two. This makes them far easier for geneticists to work with in the lab, says virologist Paul Wichgers Schreur of Wageningen University in the Netherlands. Indeed, sdAbs (also known as nanobodies, or VHHs) are being developed for a variety of applications and disease treatments, including antiviral therapies. The idea is that infected patients would be given sdAbs to bind and neutralize the virus, slowing its spread in the body.

To achieve effective virus neutralization, sdAbs tend to be combined into multimers to improve binding—as soon as one sdAb releases a target, another sdAb is close by to immediately grab hold of it. Such multimers, which are composed of either multiple copies of the same sdAB or a mixture of different ones, are typically genetically engineered—requiring them to be cloned in bacteria or yeast. But “if you want to search for the best combination” of sdAbs for binding the virus, says Wichgers Schreur, “you have to clone all the options,” which can be tedious and tricky.

To make multimeric antibody complexes to fight a pathogen, llamas are inoculated with a virus of interest 1. Researchers collect DNA from antibody-making cells in the llama’s blood and amplify it to produce single-domain antibodies (sdAbs) 2. This collection of sdAbs is screened to find those with a strong virus-binding ability 3, and those antibodies are then tagged with bacterial superglue peptides (superglues are peptide-protein partners that form irreversible bonds) 4. By mixing the glue-tagged sdAbs with scaffolds containing the glue partner proteins, researchers can combine their desired sdAbs into multimers 5, and then screen them to find the ones that best neutralize the virus 6.

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Wichgers Schreur’s team opted for a faster method: sticking various sdAbs from llamas together with bacterial superglue—a technique developed by other groups for forming irreversible bonds between special bacterial peptides and their partner proteins. The team used a variety of available peptide-protein pairs, including SpyTag-SpyCatcher and SnoopTag-SnoopCatcher.

One of the team’s test targets was Rift Valley fever virus (RVFV), which primarily affects ruminants but can be transmitted to humans, and for which there is no vaccine. They isolated sdAbs from llamas inoculated with RVFV, tagged the sdAbs with superglue peptides, then mixed different combinations of tagged sdABs together with scaffold proteins containing the corresponding catcher sequences.

The team tested these combinations in vitro to find one with potent RVFV-neutralizing ability, then turned to traditional genetic engineering to rebuild it. This enabled the researchers to modify the multimer to be more stable and less immunogenic than the original glued-together version. Infected mice treated with the multimer had reduced mortality compared with untreated mice.

While the work focused on RVFV, “[the] technologies can easily be transferred to the current COVID-19 research” or work on other viral diseases, says Edward Dolk, the CEO of a company called QVQ that develops sdABs but was not involved in the research. (eLife, 9:e52716, 2020)

Multimerization of sdAbs How it works Advantages Disadvantages Therapeutic use
Genetic fusionMultiple individual sdAb coding sequences are fused with linker sequences to create a multimer-encoding sequence that is cloned into an expression vector in bacteria or yeast.Based on a well-established technique that can be applied in most molecular biology laboratoriesEach combination of sdAbs to be tested needs to be individually cloned. Production yields of these multimers are frequently low.Yes, in clinical trials. Can be applied to patients directly and can be designed to minimize immunogenicity
Bacterial superglueEach individual sdAb is tagged with one of a series of glue peptides. These then couple to scaffold proteins containing corresponding catcher domains to form a variety of multimers.Reserachers can assemble and test combinations of sdAbs rapidly. Production of individual building blocks is generally efficient.Technique not yet optimal. Coupling not always 100 percent. Bacterial superglue proteins are immunogenic.
Not directly. The selected multimer should be reformatted into a human antibody–like molecule.

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  54. I do agree with all of the ideas you have presented in your post. They are really convincing and will certainly work. Still, the posts are too short for novices. Could you please extend them a bit from next time? Thanks for the post.

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