Angiopep-2 fused VHH E12 and C7C18 peptides efficiently cross BBB model and neutralize SARS-CoV-2

Therapy for SARS-CoV-2 central nervous system (CNS) infection currently focuses on managing neuroinflammation and associated damage, rather than directly targeting the virus. A major hurdle for developing specific antiviral therapeutics for CNS infections is the limited permeability of drugs across the blood-brain barrier (BBB). This physiological barrier restricts the entry of most large molecules and many small molecules into the brain, leaving the CNS vulnerable to viral replication without direct antiviral intervention. This research addresses this critical gap by developing novel antiviral agents capable of traversing the BBB.

Researchers developed a pipeline to select single variable domain of llama heavy chain antibodies (VHHs) and 7-mer cyclic peptides (C7C) from phage-display libraries that bind to the SARS-CoV-2 spike protein. Soluble VHH and C7C peptides were overexpressed in E. coli SHuffle Express to ensure proper disulfide bond formation. These candidates were rigorously tested for pseudovirus neutralization, cell toxicity, and hemocompatibility. The most promising candidates, VHH E12 and C7C18, were then genetically fused with the CNS-homing peptide Angiopep-2 and re-evaluated for viral neutralization and in vitro BBB crossing efficiency.

The selected VHH E12 and C7C18 candidates demonstrated potent neutralization of live SARS-CoV-2 in a plaque reduction neutralization test, showing EC50 values of 0.045 μg/mL and 0.01 μg/mL, respectively. However, these original peptides failed to cross the in vitro blood-brain barrier model effectively. Upon fusion with Angiopep-2, the constructs exhibited significantly enhanced BBB permeability.

VHH E12-Angiopep-2 showed a 5.4-fold increase in BBB crossing, while C7C18-Angiopep-2 demonstrated an even more substantial 11.2-fold increase in crossing efficiency. Crucially, both fusion constructs maintained their robust ability to neutralize the virus, indicating that the Angiopep-2 modification did not compromise their antiviral activity. The C7C18-Angiopep-2 molecule, in particular, was highlighted for its exceptional potential.