Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and older people. Current RSV subunit vaccines are based on a fusion protein that is stabilized in the prefusion conformation and linked to a heterologous foldon trimerization domain to obtain a prefusion F (preF) trimer. Here we show that current RSV vaccines induce undesirable anti-foldon antibodies in non-human primates, mice and humans. To overcome this, we designed a foldon-free RSV preF trimer by elucidating the structural basis of trimerization-induced preF destabilization through molecular dynamics simulations and by introducing amino acid substitutions that negate hotspots of charge repulsion. The highly stable prefusion conformation was validated using antigenic and cryo-electron microscopy analysis. The preF is immunogenic and protective in naive mouse models and boosts neutralizing antibody titres in RSV-pre-exposed mice and non-human primates, while achieving similar titres to approved RSV vaccines in mice. This stable preF design is a promising option as a foldon-independent candidate for a next-generation RSV vaccine immunogen.

呼吸道合胞病毒 （RSV） 是婴儿和老年人严重呼吸系统疾病的主要原因。目前的 RSV 亚单位疫苗基于一种融合蛋白，该融合蛋白稳定在融合前构象中，并与异源折叠三聚体化结构域相连，以获得融合前的 F （preF） 三聚体。在这里，我们表明目前的 RSV 疫苗在非人灵长类动物、小鼠和人类中诱导不需要的抗折叠抗体。为了克服这个问题，我们设计了一种无折叠的 RSV preF 三聚体，通过分子动力学模拟阐明了三聚体化诱导的 preF 不稳定的结构基础，并通过引入抵消电荷排斥热点的氨基酸取代。使用抗原和冷冻电子显微镜分析验证了高度稳定的预融合构象。preF 在幼稚小鼠模型中具有免疫原性和保护性，可提高 RSV 预暴露小鼠和非人灵长类动物的中和抗体滴度，同时在小鼠中实现与已批准的 RSV 疫苗相似的滴度。这种稳定的 preF 设计作为下一代 RSV 疫苗免疫原的不依赖折叠的候选者是一个很有前途的选择。