Anti-SARS-CoV-2 VHH and C7C peptide fused with Angiopep-2 efficiently traverse blood-brain barrier model and...

The therapy for SARS-CoV-2-related CNS infection relies on treating neuroinflammation and associated damage, rather than a specific antiviral. The lack of specific antiviral therapeutics is mainly attributed to the limited permeability of drugs across the blood-brain barrier.

In this paper, we present a detailed pipeline for selecting VHHs (single variable domain of llama heavy chain antibody) and 7-mer cyclic peptides (C7C) from phage-display libraries that bind to the spike protein and SARS-CoV-2 virion. Soluble VHH and C7C peptides overexpressed in the E. coli SHuffle Express, which enables the formation of the disulfide bond essential for proper VHH and C7C folding and function, were purified and rigorously tested for their capacity to neutralize pseudovirus, cell toxicity, hemocompatibility, etc. The best VHH and C7C candidates with favorable attributes were then fused with the CNS-homing peptide Angiopep-2 and thoroughly assessed for the ability to neutralize the virus along with other safety attributes mentioned earlier.

The best VHH and C7C candidates, VHH _E12 and C7C₁₈, tested for their ability to neutralize live virus in a plaque reduction neutralization test, showed EC₅₀ 0.045 μg/mL and 0.01 μg/mL, respectively. Both VHH _E12 and C7C₁₈, however, fail to cross the blood-brain barrier in vitro, thus were fused with CNS-homing peptide Angiopep-2. VHH _E12 -Angiopep-2 and C7C₁₈-Angiopep-2 fusion constructs showed increased crossing across the blood-brain barrier by 5.4-fold and 11.2-fold, respectively, while maintaining their ability to neutralize the virus. Molecules generated here, particularly C7C₁₈-Angiopep-2, hold merit for further in vivo testing and preclinical studies.