Structure of the rabies virus glycoprotein trimer bound to a pre-fusion specific neutralizing antibody

Rabies infection is nearly 100% lethal if untreated and kills over 50,000 people annually, many of them children. Existing rabies vaccines target the rabies virus glycoprotein (RABV-G) but generate short-lived immune responses, likely because the protein is heterogeneous under physiological conditions. Here, we report the 3.39Å cryo-EM structure of trimeric, pre-fusion RABV-G complexed with RVA122, a potently neutralizing human antibody. RVA122 binds to a quaternary epitope at the top of RABV-G, bridging domains and stabilizing RABV-G protomers in a prefusion state. RABV-G trimerization involves side-to-side interactions between the central α-helix and adjacent loops, rather than contacts between central helices, and interactions among the fusion loops at the glycoprotein base. These results provide a basis to develop improved rabies vaccines based on RABV-G stabilized in the prefusion conformation.

Virus-like vesicles based on SFV-containing rabies virus glycoprotein make a safe and efficacious rabies vaccine candidate in a mouse model.

Rabies is a fatal zoonosis causing encephalitis in mammals, and vaccination is the most effective method to control and eliminate rabies. Virus-like vesicles (VLVs), which are characterized as infectious, self-propagating membrane-enveloped particles composed of only Semliki Forest virus (SFV) replicase and vesicular stomatitis virus glycoprotein (VSV-G), have been proven safe and efficient as vaccine candidates. However, previous studies showed that VLVs containing rabies virus glycoprotein (RABV-G) grew at relatively low titers in cells, impeding their potential use as a rabies vaccine. In this study, we constructed novel VLVs by transfection of a mutant SFV RNA replicon encoding RABV-G. We found these VLVs could self-propagate efficiently in cell culture and could evolve to high titers (approximately 10 8 FFU/ml) by extensive passaging 25 times in BHK-21 cells. Furthermore, we found that the evolved amino acid change in SFV nsP1 at positions 470 and 482 was critical for this high-titer phenotype. Remarkably, VLVs could induce robust type I IFN expression in BV2 cells and were highly sensitive to IFN-α. We found that direct inoculation of VLVs into the mouse brain caused lesser body weight loss, mortality and neuroinflammation compared with RABV vaccine strain. Finally, it could induce increased generation of germinal centre (GC) B cells, plasma cells (PCs) and virus-neutralizing antibodies (VNAs), as well as provide protection against virulent RABV challenge in immunized mice. This study demonstrated that VLVs containing RABV-G could proliferate in cells and were highly evolvable, revealing the feasibility of developing an economic, safe and efficacious rabies vaccine. IMPORTANCE VLVs have been shown to represent a more versatile and superior vaccine platform. In previous studies, VLVs containing the Semliki Forest Virus replicase (SFV nsP1-4) and rabies virus glycoprotein (RABV-G) grew to relatively low titers in cells. In our study, we not only succeeded in generating VLVs that proliferate in cells and stably express RABV-G, the VLVs that evolved grew to higher titers reaching 10 8 FFU/ml. We also found that nucleic acid changes at positions 470 and 482 in nsP1 were vital for this high-titer phenotype. Moreover, the VLVs that evolved in our studies were highly attenuated in mice, induced potent immunity and protected mice from lethal RABV infection. Collectively, our study showed that high titers of VLVs containing RABV-G were achieved demonstrating that these VLVs could be an economical, safe, and efficacious rabies vaccine candidate.