In a recent study published in PLOS Pathogens, researchers screened a library of phages encoding biosynthetic proteins called αReps. They pursued αReps that might block the interaction between receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein with the host’s angiotensin-converting enzyme 2 (ACE2) receptors.

Study: Biosynthetic proteins targeting the SARS-CoV-2 spike as anti-virals. Image Credit: creativeneko/Shutterstock

Background

It’s crucial to develop efficient antiviral strategies against SARS-CoV-2 to mitigate and control the coronavirus disease 2019 (COVID-19) crisis which has already resulted in over six million deaths globally. A technique to fight the spread of all respiratory viruses can be needed to combat future pandemics.

SARS-CoV-2 initiates infection within the nasal cavity, replicates within the olfactory epithelia and lower respiratory tract (URT), after which reaches the lower respiratory tract (LRT), where it establishes infection. Because it causes massive damage within the olfactory epithelia, an antiviral blocking virus multiplication within the nose and URT could offer immense therapeutic advantages and even provide prophylactic protection.

Some human neutralizing monoclonal immunoglobulin G (IgG) antibodies inhibit SARS-CoV-2 infection within the nose and URT; nonetheless, their use is proscribed. Furthermore, their firmness upon nebulization and aerosolization is poor. They require injection and can’t be taken orally or by spray.

αReps are 31-amino acids (AAs) long thermostable proteins commonly present in eukaryotes and prokaryotes, including thermophiles. Sequences of homologous αReps form a contrasted sequence profile, with most positions occupied by conserved AAs and the remaining positions generating a versatile binding surface. Scientists have assembled an enormous αRep library with an unlimited collection of disparate protein targets, particularly, to interact and deleteriously interfere with virus maturation (e.g., against human immunodeficiency virus nucleocapsid protein).

The SARS-CoV-2 spike (S) protein, just like the S proteins of all of the coronaviruses, harbors RBD. RBD is a goal to discover ligands that block interaction with the host-cell receptors. Nonetheless, SARS-CoV-2 evolution has led to the emergence of explicit escape mutations within the RBD, making the present variable domain of heavy-chain antibodies (VHH)-based treatments much less effective.

In regards to the study

In the current study, researchers developed an αRep series specific to the RBD of the SARS-CoV-2 S, which could easily adapt to work stably against SARS-CoV-2 variants at a low price. The researchers used a previously established method to construct the αRep library that relied on the polymerization of synthetic microgenes corresponding to individual HEAT-like repeats. They used the surface of M13-derived filamentous phages to specific the αRep proteins.

They cloned αRep genes encoding the RBD binders within the bacterial expression vector pQE81 and used the resulting plasmids for transforming Rosetta cells. The library was estimated to contain 1.7 x 109 independent clones.

Further, the researchers performed binding kinetics experiments and used biolayer interferometry for affinity determination. Moreover, they performed competition assays between αReps using biotinylated SARS-CoV-2 RBD loaded on streptavidin (SA) biosensors.

Study findings

The C2 and F9 αReps displayed SARS-CoV-2 RBD affinity within the nanomolar (nM) range. The F9-C2 heterodimer and homotrimeric C2-foldon showed higher neutralization activity against SARS-CoV-2 than the parental αReps. They exhibited a half-maximal inhibitory concentration (IC50) of three to 12 nM.

Furthermore, F9-C2 and C2-foldon helped efficient neutralization of a wide selection of SARS-CoV-2 variants, including Omicron. This feature could be as a result of the inherent high affinity of the αRep subunits for the RBD and their multimerization, which allowed lesser dependence on AA substitutions within the goal.

Competitive binding assays showed that αReps neutralized SARS-CoV-2 through different mechanisms, with C2 binding a site distant from the receptor binding motif to ACE2 and overlapping the VHH H11D4 epitope. Contrastingly, F9 competed with ACE2 and VHH-72 for binding the RBD. Within the hamster model, treatment with C2 and F9 αReps induced a discount within the nasal viral load (the first replication site of SARS-CoV-2), plus a decline of all of the inflammation markers of infection.

Conclusions

With optimization in binder selection and efforts to stabilize αReps within the nasal cavity, these biosynthetic proteins and their derivates could emerge as promising and versatile neutralizing binders targeting the SARS-CoV-2 S protein. They might even be a low-cost potential treatment for emerging respiratory viruses. Nonetheless, future studies should address the immunogenicity problems with αReps. Notably, they’re relatively small-sized (e.g., the C2 αRep is eighteen.5 kDa), hence, their high solubility, stability, and association through flexible linkers end in low immunogenic activity, which, in turn, hampers the induction of antagonistic effects when delivered within the nasal cavity.