Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in infants, and an effective vaccine is not yet available. We previously generated an RSV live-attenuated vaccine (LAV) candidate, DB1, which was attenuated by a low-fusion subgroup B F protein (BAF) and codon-deoptimized nonstructural protein genes. DB1 was immunogenic and protective in cotton rats but lacked thermostability and stability of the prefusion conformation of F compared to strains with the line19F gene. We hypothesized that substitution of unique residues from the thermostable A2-line19F strain could thermostabilize DB1 and boost its immunogenicity. We therefore substituted 4 unique line19F residues into the BAF protein of DB1 by site-directed mutagenesis and rescued the recombinant virus, DB1-QUAD. Compared to DB1, DB1-QUAD had improved thermostability at 4°C and higher levels of prefusion F as measured by enzyme-linked immunosorbent assays (ELISAs). DB1-QUAD was attenuated in normal human bronchial epithelial cells, in BALB/c mice, and in cotton rats but grew to wild-type titers in Vero cells. In mice, DB1-QUAD was highly immunogenic and generated significantly higher neutralizing antibody titers to a panel of RSV A and B strains than did DB1. DB1-QUAD was also efficacious against wild-type RSV challenge in mice and cotton rats. Thus, substitution of unique line19F residues into RSV LAV DB1 enhanced vaccine thermostability, incorporation of prefusion F, and immunogenicity and generated a promising vaccine candidate that merits further investigation.

IMPORTANCE We boosted the thermostability and immunogenicity of an RSV live-attenuated vaccine candidate by substituting 4 unique residues from the RSV line19F protein into the F protein of the heterologous vaccine strain DB1. The resultant vaccine candidate, DB1-QUAD, was thermostable, attenuated in vivo, highly immunogenic, and protective against RSV challenge in mice and cotton rats.

呼吸道合胞病毒（RSV）是婴儿下呼吸道感染的主要原因，目前尚无有效的疫苗。我们先前生成了一种RSV减毒活疫苗（LAV）候选物DB1，它被低融合亚组BF蛋白（BAF）和密码子去优化的非结构蛋白基因减毒。DB1在棉鼠中具有免疫原性和保护性，但与带有line19F基因的菌株相比，F1的热稳定性和融合前构象缺乏稳定性。我们假设从热稳定的A2-line19F菌株中唯一残基的取代可以使DB1热稳定并增强其免疫原性。因此，我们通过定点诱变将4个独特的line19F残基替换为DB1的BAF蛋白，并拯救了重组病毒DB1-QUAD。与DB1相比，通过酶联免疫吸附测定（ELISA），DB1-QUAD在4°C时具有更高的热稳定性，并具有更高的预融合F水平。在正常人支气管上皮细胞，BALB / c小鼠和棉鼠中，DB1-QUAD减毒，但在Vero细胞中生长至野生型滴度。在小鼠中，DB1-QUAD具有高度的免疫原性，与一组RSV A和B株相比，产生的中和抗体效价明显高于DB1。DB1-QUAD对小鼠和棉鼠的野生型RSV攻击也有效。因此，将独特的line19F残基替换为RSV LAV DB1可增强疫苗的热稳定性，掺入融合前F和提高免疫原性，并产生了值得进一步研究的有前途的候选疫苗。在正常人支气管上皮细胞，BALB / c小鼠和棉鼠中，DB1-QUAD减毒，但在Vero细胞中生长至野生型滴度。在小鼠中，DB1-QUAD具有高度的免疫原性，与一组RSV A和B株相比，产生的中和抗体效价明显高于DB1。DB1-QUAD对小鼠和棉鼠的野生型RSV攻击也有效。因此，将独特的line19F残基替换为RSV LAV DB1可增强疫苗的热稳定性，掺入融合前F和提高免疫原性，并产生了值得进一步研究的有前途的候选疫苗。在正常人支气管上皮细胞，BALB / c小鼠和棉鼠中，DB1-QUAD减毒，但在Vero细胞中生长至野生型滴度。在小鼠中，DB1-QUAD具有高度的免疫原性，与一组RSV A和B株相比，产生的中和抗体效价明显高于DB1。DB1-QUAD对小鼠和棉鼠的野生型RSV攻击也有效。因此，将独特的line19F残基替换为RSV LAV DB1可增强疫苗的热稳定性，掺入融合前F和提高免疫原性，并产生了值得进一步研究的有前途的候选疫苗。DB1-QUAD具有高度的免疫原性，与DB1相比，对一组RSV A和B菌株产生的中和抗体效价明显更高。DB1-QUAD对小鼠和棉鼠的野生型RSV攻击也有效。因此，将独特的line19F残基替换为RSV LAV DB1可增强疫苗的热稳定性，掺入融合前F和提高免疫原性，并产生了值得进一步研究的有前途的候选疫苗。DB1-QUAD具有高度的免疫原性，与DB1相比，对一组RSV A和B菌株产生的中和抗体效价明显更高。DB1-QUAD对小鼠和棉鼠的野生型RSV攻击也有效。因此，将独特的line19F残基替换为RSV LAV DB1可增强疫苗的热稳定性，掺入融合前F和提高免疫原性，并产生了值得进一步研究的有前途的候选疫苗。

重要我们通过将RSV line19F蛋白的4个独特残基替换为异源疫苗株DB1的F蛋白，提高了RSV减毒活疫苗候选物的热稳定性和免疫原性。所得的候选疫苗DB1-QUAD具有热稳定性，体内减毒作用，高度免疫原性，并能抵抗小鼠和棉鼠的RSV攻击。