A high-throughput screening assay to identify inhibitory antibodies targeting alphavirus release

Abstract

Background Several studies have demonstrated neutralizing antibodies to be highly effective against alphavirus infection in animal models, both prophylactically and remedially. In most studies, neutralizing antibodies have been evaluated for their ability to block viral entry in vitro but recent evidence suggests that antibody inhibition through other mechanisms, including viral budding/release, significantly contributes to viral control in vivo for a number of alphaviruses. Results We describe a BSL-2, cell-based, high-throughput screening system that specifically screens for inhibitors of alphavirus egress using chikungunya virus (CHIKV) and Mayaro virus (MAYV) novel replication competent nano-luciferase (nLuc) reporter viruses. Screening of both polyclonal sera and memory B-cell clones from CHIKV immune individuals using the optimized assay detected several antibodies that display potent anti-budding activity. Conclusions We describe an “anti-budding assay” to specifically screen for inhibitors of viral egress using novel CHIKV and MAYV nLuc reporter viruses. This BSL-2 safe, high-throughput system can be utilized to explore neutralizing “anti-budding” antibodies to yield potent candidates for CHIKV and MAYV therapeutics and prophylaxis.

Chikungunya Virus Release is Reduced by TIM-1 Receptors Through Binding of Envelope Phosphatidylserine

“…Surprisingly, CHIKV displayed the highest levels of release in BSR-T7/5 cells. Similar findings were previously evidenced with BHK cells in Ramjag, et al, 2022 (22). We observed an inverse correlation between PC:PE:PS liposome binding and particle release in Vero, VeroS, VeroΔTIM/AXL, Aag2, and BSR-T7/5 cells (Figure 6D).…”

“…To more readily quantify CHIKV viral release efficiency, we cloned nano-luciferase (NLuc) to the N-terminus of the E2 glycoprotein as previously described (Figure 1B) (22). The recombinant virus contains NLuc in the virion, therefore viral particles can be readily quantified using a standard luminescence assay.…”