Prefusion-Stabilized Lassa Virus Trimer Identifies Neutralizing Nanobodies and Reveals an Apex-Situated Site of Vulnerability

Lassa virus (LASV) is responsible for 100,000-300,000 zoonotic infections annually and poses a threat to public health. Development of antibody-based therapeutics or vaccines has been challenging because neutralizing antibodies – even among Lassa hemorrhagic fever survivors – are generally of low titer, and the target of neutralizing antibodies, the trimeric glycoprotein complex (GPC), a type 1-fusion machine with GP1 and GP2 subunits, has been difficult to produce. Here, we use structure-based design to obtain a soluble LASV GPC by engineering an inter-protomer disulfide (R207GCGP1-L326CGP2) and appending the T4-fibritin trimerization domain. We verified the antigenicity of this prefusion-stabilized LASV GPC against a panel of human antibodies and used electron microscopy (EM) to confirm its trimeric association. We panned the prefusion-stabilized LASV GPC against single domain ‘nanobody’ libraries and identified one of camel origin, which we named D5, which bound GPC with 27 nM affinity and neutralized the Josiah strain of LASV with an IC50 of 12 µg/ml when formatted into a bivalent IgG2a context. The cryo-EM structure of a ternary complex of the D5 nanobody, the antigen-binding fragment of human antibody 8.11G, and LASV GPC revealed D5 to recognize a site-of-vulnerability at the trimer apex. The recognized site appeared to be specific to GPC lacking cleavage of between GP1 and GP2 subunits. Collectively, our findings suggest that GPC-cleavage intermediates may be targets for LASV neutralization and define an apex-situated site of vulnerability for vaccine development. Significance Lassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a biothreat to the world. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into requirements for antibody-mediated neutralization of LASV, we developed a prefusion-stabilized LASV glycoprotein trimer (GPC), panned it against phage libraries comprised single-domain antibodies or nanobodies from shark and camel, and identified one, D5, which – when placed into bivalent IgG2a context – could neutralize LASV. Cryo-EM analysis revealed D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. We propose this apex-situated site to be an attractive target for LASV vaccine and therapeutic development. ### Competing Interest Statement The authors have declared no competing interest.