State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural Universitygrid.35155.37, Wuhan, China.
Key Laboratory of Preventive Veterinary Medicine of Hubei Province, Huazhong Agricultural Universitygrid.35155.37, Wuhan, China.
J Virol. 2021 Sep 27;95(20):e0079021. doi: 10.1128/JVI.00790-21. Epub 2021 Aug 4.
Rabies is a fatal zoonosis that causes 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 that these VLVs could self-propagate efficiently in cell culture and could evolve to high titers (approximately 10 focus-forming units [FFU]/ml) by extensive passaging 25 times in BHK-21 cells. Furthermore, we found that the evolved amino acid changes in SFV nonstructural protein 1 (nsP1) at positions 470 and 482 was critical for this high-titer phenotype. Remarkably, VLVs could induce robust type I interferon (IFN) expression in BV2 cells and were highly sensitive to IFN-α. We found that direct inoculation of VLVs into the mouse brain caused reduced body weight loss, mortality, and neuroinflammation compared with the RABV vaccine strain. Finally, it could induce increased generation of germinal center (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. 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 to nsP4) 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, but the VLVs that evolved grew to higher titers, reaching 10 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.
狂犬病是一种致命的人畜共患病，会导致哺乳动物脑炎，而疫苗接种是控制和消除狂犬病的最有效方法。病毒样囊泡（VLVs）是一种具有传染性、自我复制的膜包裹颗粒，仅由 Semliki Forest 病毒（SFV）复制酶和水疱性口炎病毒糖蛋白（VSV-G）组成，已被证明是安全有效的疫苗候选物。然而，先前的研究表明，含有狂犬病病毒糖蛋白（RABV-G）的 VLVs 在细胞中的生长滴度相对较低，这阻碍了它们作为狂犬病疫苗的潜在用途。在这项研究中，我们通过转染编码 RABV-G 的突变 SFV RNA 复制子构建了新型 VLVs。我们发现，这些 VLVs 可以在细胞培养中高效自我复制，并通过在 BHK-21 细胞中广泛传代 25 次，进化到高滴度（约 10 个焦点形成单位[FFU]/ml）。此外，我们发现 SFV 非结构蛋白 1（nsP1）位置 470 和 482 的进化氨基酸变化对于这种高滴度表型至关重要。值得注意的是，VLVs 可以在 BV2 细胞中诱导强烈的 I 型干扰素（IFN）表达，并且对 IFN-α 高度敏感。我们发现，与 RABV 疫苗株相比，直接将 VLVs 接种到小鼠大脑中会导致体重减轻、死亡率和神经炎症减少。最后，它可以诱导生发中心（GC）B 细胞、浆细胞（PCs）和病毒中和抗体（VNAs）的大量产生，并为免疫小鼠提供针对强毒 RABV 攻击的保护。这项研究表明，含有 RABV-G 的 VLVs 可以在细胞中增殖并且高度可进化，这表明开发经济、安全、有效的狂犬病疫苗是可行的。VLVs 已被证明是一种更通用和优越的疫苗平台。在先前的研究中，含有 Semliki Forest 病毒复制酶（SFV nsP1 到 nsP4）和狂犬病病毒糖蛋白（RABV-G）的 VLVs 在细胞中生长到相对较低的滴度。在我们的研究中，我们不仅成功地生成了在细胞中增殖并稳定表达 RABV-G 的 VLVs，而且进化的 VLVs 生长到更高的滴度，达到 10 FFU/ml。我们还发现 nsP1 位置 470 和 482 的核酸变化对于这种高滴度表型至关重要。此外，在我们的研究中进化的 VLVs 在小鼠中高度减毒，诱导强烈的免疫反应，并保护小鼠免受致命的 RABV 感染。总的来说，我们的研究表明，实现了含有 RABV-G 的 VLVs 的高滴度，表明这些 VLVs 可以成为一种经济、安全、有效的狂犬病疫苗候选物。
