Human respiratory syncytial virus (RSV) is the leading cause of pediatric bronchiolitis and hospitalizations. RSV can also cause severe complications in elderly and immunocompromised individuals. There is no licensed vaccine. We previously generated a parainfluenza virus 5 (PIV5)-vectored vaccine candidate expressing the RSV fusion protein (F) that was immunogenic and protective in mice. In this work, our goal was to improve the original vaccine candidate by modifying the PIV5 vector or by modifying the RSV F antigen. We previously demonstrated that insertion of a foreign gene at the PIV5 small hydrophobic (SH)–hemagglutinin-neuraminidase (HN) junction or deletion of PIV5 SH increased vaccine efficacy. Additionally, other groups have demonstrated that antibodies against the prefusion conformation of RSV F have more potent neutralizing activity than antibodies against the postfusion conformation. Therefore, to improve on our previously developed vaccine candidate, we inserted RSV F at the PIV5 SH-HN gene junction or used RSV F to replace PIV5 SH. We also engineered PIV5 to express a prefusion-stabilized F mutant. The candidates were tested in BALB/c mice via the intranasal route and induced both humoral and cell-mediated immunity. They also protected against RSV infection in the mouse lung. When they were administered intranasally or subcutaneously in cotton rats, the candidates were highly immunogenic and reduced RSV loads in both the upper and lower respiratory tracts. PIV5-RSV F was equally protective when administered intranasally or subcutaneously. In all cases, the prefusion F mutant did not induce higher neutralizing antibody titers than wild-type F. These results show that antibodies against both pre- and postfusion F are important for neutralizing RSV and should be considered when designing a vectored RSV vaccine. The findings also that indicate PIV5-RSV F may be administered subcutaneously, which is the preferred route for vaccinating infants, who may develop nasal congestion as a result of intranasal vaccination.

IMPORTANCE Despite decades of research, human respiratory syncytial virus (RSV) is still a major health concern for which there is no vaccine. A parainfluenza virus 5-vectored vaccine expressing the native RSV fusion protein (F) has previously been shown to confer robust immunity against RSV infection in mice, cotton rats, and nonhuman primates. To improve our previous vaccine candidate, we developed four new candidates that incorporate modifications to the PIV5 backbone, replace native RSV F with a prefusion-stabilized RSV F mutant, or combine both RSV F and PIV5 backbone modifications. In this work, we characterized the new vaccine candidates and tested their efficacies in both murine and cotton rat models of RSV infection. Most importantly, we found that PIV5-based RSV vaccine candidates were efficacious in preventing lower respiratory tract infection as well as in reducing the nasal viral load when administered via the subcutaneous route.

人类呼吸道合胞病毒 (RSV) 是小儿细支气管炎和住院治疗的主要原因。 RSV 还会导致老年人和免疫功能低下的人出现严重并发症。没有获得许可的疫苗。我们之前生成了一种副流感病毒 5 (PIV5) 载体候选疫苗，表达 RSV 融合蛋白 (F)，该疫苗在小鼠中具有免疫原性和保护性。在这项工作中，我们的目标是通过修饰 PIV5 载体或修饰 RSV F 抗原来改进原始候选疫苗。我们之前证明，在 PIV5 小疏水性（SH）-血凝素-神经氨酸酶（HN）连接处插入外源基因或删除 PIV5 SH 可以提高疫苗功效。此外，其他研究小组已经证明，针对 RSV F 融合前构象的抗体比针对融合后构象的抗体具有更有效的中和活性。因此，为了改进我们之前开发的候选疫苗，我们在 PIV5 SH-HN 基因连接处插入 RSV F 或使用 RSV F 代替 PIV5 SH。我们还设计了 PIV5 来表达融合前稳定的 F 突变体。这些候选药物通过鼻内途径在 BALB/c 小鼠中进行了测试，并诱导了体液和细胞介导的免疫。它们还可以保护小鼠肺部免受 RSV 感染。当对棉鼠进行鼻内或皮下注射时，候选药物具有高度免疫原性，并降低了上呼吸道和下呼吸道中的 RSV 负荷。当鼻内或皮下给药时，PIV5-RSV F 具有同样的保护作用。在所有情况下，融合前的 F 突变体并未诱导比野生型 F 更高的中和抗体滴度。 这些结果表明，针对融合前和融合后 F 的抗体对于中和 RSV 很重要，在设计载体化 RSV 疫苗时应予以考虑。研究结果还表明，PIV5-RSV F 可以皮下注射，这是婴儿疫苗接种的首选途径，婴儿可能因鼻内疫苗接种而出现鼻塞。

重要性尽管经过数十年的研究，人类呼吸道合胞病毒 (RSV) 仍然是一个主要的健康问题，而且还没有疫苗。先前已证明表达天然 RSV 融合蛋白 (F) 的副流感病毒 5 载体疫苗可赋予小鼠、棉鼠和非人灵长类动物针对 RSV 感染的强大免疫力。为了改进我们之前的候选疫苗，我们开发了四种新的候选疫苗，它们结合了对 PIV5 主链的修饰，用融合前稳定的 RSV F 突变体取代天然 RSV F，或者结合了 RSV F 和 PIV5 主链修饰。在这项工作中，我们对新候选疫苗进行了表征，并在小鼠和棉鼠 RSV 感染模型中测试了它们的功效。最重要的是，我们发现基于 PIV5 的 RSV 候选疫苗在通过皮下途径给药时可有效预防下呼吸道感染以及减少鼻腔病毒载量。