Abstract The lethal rabies encephalitis diseases are caused by members of the zoonotic lyssavirus genus. The high genetic diversity separates lyssavirus members into three phylogroups, while cross-phylogroup (especially to phylogroups 2 and 3) protection has not been established by current post-exposure prophylaxis (PEP) or antibody therapeutics. To achieve more broadly effective countermeasures to rabies disease worldwide and to prepare for the emergence of new lyssaviruses, effective therapeutic agents against all phylogroups are necessary. Lyssavirus G glycoprotein is the sole antibody target on the virion surface, which adopt distinct conformations between pre- and post- fusion states as other class-III viral fusion machineries. Domain-III of lyssavirus G glycoprotein encompasses a large neutralizing antibody-accessible surface in the native state. Sequence analysis on domain-III revealed several conserved patches across all phylogroups, which could potentially serve as target epitopes for pan-lyssavirus neutralizers. Antigen-specific nanobodies have been considered as promising therapeutic agents against various infectious diseases and non-infectious diseases, which have the advantage in binding compact and hidden epitopes that are out of reach for conventional antibodies. We hypothesized that by focusing immune recognition on the lyssavirus G domain-III conserved epitopes, we can identify pan-lyssavirus neutralizing nanobodies for immunotherapeutics. In this study, we propose two specific aims: (1) to use a structure-based and antibody-guided approach to design and characterize the antigenicity of lyssavirus glycoprotein domain-III, a site of viral vulnerability targeted by several broadly neutralizing antibodies, for revelation of the antibody neutralization mechanism; (2) to identify and characterize pan-lyssavirus neutralizing nanobodies for therapeutics by focusing immune recognition on lyssavirus G domain III conserved epitopes.

Narrative Zoonotic lyssaviruses causing acute rabies encephalitis diseases are responsible for the death of over 59,000 people each year worldwide. Current treatments and vaccines are effective only towards a subset of lyssavirus members due to the high genetic and antigenic diversity. We propose to target a conserved site of viral vulnerability to develop pan-lyssavirus nanobody therapeutics against both circulating and emerging lyssaviruses.