Rostad Christina A, Stobart Christopher C, Todd Sean O, Molina Samuel A, Lee Sujin, Blanco Jorge C G, Moore Martin L
Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia, USA.
Children's Healthcare of Atlanta, Atlanta, Georgia, USA.
J Virol. 2018 Feb 26;92(6). doi: 10.1128/JVI.01568-17. Print 2018 Mar 15.
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. 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 , 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病因，目前尚无有效的疫苗。我们之前研发了一种RSV减毒活疫苗（LAV）候选株DB1，它通过低融合亚组B F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，但与具有line19F基因的毒株相比，缺乏热稳定性和F蛋白预融合构象的稳定性。我们推测，用来自热稳定的A2-line19F毒株的独特残基进行替换可以使DB1具有热稳定性并增强其免疫原性。因此，我们通过定点诱变将4个独特的line19F残基替换到DB1的BAF蛋白中，并拯救出重组病毒DB1-QUAD。与DB1相比，DB1-QUAD在4°C时具有更好的热稳定性，通过酶联免疫吸附测定（ELISA）测量，其预融合F水平更高。DB1-QUAD在正常人支气管上皮细胞、BALB/c小鼠和棉鼠中减毒，但在Vero细胞中生长至野生型滴度。在小鼠中，DB1-QUAD具有高度免疫原性，与DB1相比，对一组RSV A和B毒株产生的中和抗体滴度显著更高。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残基替换到RSV LAV DB1中可增强疫苗的热稳定性、预融合F的掺入以及免疫原性，并产生了一个有前景的疫苗候选株，值得进一步研究。我们通过将RSV line19F蛋白的4个独特残基替换到异源疫苗株DB1的F蛋白中，提高了RSV减毒活疫苗候选株的热稳定性和免疫原性。所得的疫苗候选株DB1-QUAD具有热稳定性、减毒、高度免疫原性，并且对小鼠和棉鼠的RSV攻击具有保护作用。
