Protective antigenic sites in respiratory syncytial virus G attachment protein outside the central conserved and cysteine noose domains

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract disease in infants. Previously, we elucidated the antibody repertoire following primary RSV infection in infants. Whole genome-fragment phage display libraries (GFPDL) expressing linear and conformational epitopes from RSV bound 100-fold more phages within attachment protein (G) following primary RSV infection. The G-reactive epitopes spanned the N- and C-termini of G ectodomain, in addition to the central conserved domain (CCD). In the current study, we examined the contribution of antigenic regions of G outside of the CCD to RSV-specific immunity. We evaluated the immunogenicity, neutralization and protective efficacy of all RSV-G antigenic sites identified following primary RSV infection using recombinant E. coli expressed G ectodomain (REG), CCD-deleted G ectodomain (REG ΔCCD), N- and C-terminal G subdomains, and antigenic site peptides. The REG ΔCCD, N- and C-terminal subdomains and peptides generated antibody titers in rabbits and mice that bound fully glycosylated Recombinant Mammalian expressed G ectodomain (RMG) and intact RSV virion particles but minimal in vitro neutralization titers compared with the intact G ectodomain. Vaccinated mice were challenged intranasally with RSV-A2 Line 19F. Viral replication in nasal cavity and lungs was significantly reduced in vaccinated animals compared to unimmunized controls. Control of viral loads post-RSV challenge correlated with serum antibody binding to the virus particles. In addition, very low Th2/Th1 cytokine ratios were found in the lungs of REG ΔCCD vaccinated mice after challenge. These data demonstrate the presence of multiple protective sites in RSV G protein outside of the CCD that could contribute to the development of a bacterially produced unglycosylated G protein as safe and protective vaccine against RSV disease.

呼吸道合胞病毒（Respiratory syncytial virus, RSV）是导致婴幼儿罹患下呼吸道疾病的主要病原体。此前本团队已阐明婴幼儿原发性RSV感染后的抗体谱（antibody repertoire）特征。表达RSV线性与构象表位的全基因组片段噬菌体展示文库（Whole genome-fragment phage display libraries, GFPDL），在原发性RSV感染后结合附着蛋白（G）的噬菌体丰度提升100倍。G蛋白反应性表位不仅覆盖G蛋白胞外域的中央保守结构域（central conserved domain, CCD），还延伸至其N端与C端区域。在本研究中，我们探究了CCD之外的G蛋白抗原区域对RSV特异性免疫的贡献。我们采用大肠杆菌重组表达的G蛋白胞外域（recombinant E. coli expressed G ectodomain, REG）、缺失CCD的G蛋白胞外域（CCD-deleted G ectodomain, REG ΔCCD）、G蛋白N端与C端亚结构域以及抗原位点肽，对原发性RSV感染后鉴定出的所有RSV-G抗原位点的免疫原性、中和活性与保护效力进行了评估。结果显示，REG ΔCCD、G蛋白N端与C端亚结构域及抗原位点肽可在兔与小鼠体内诱导产生结合完全糖基化的哺乳动物重组表达G蛋白胞外域（fully glycosylated Recombinant Mammalian expressed G ectodomain, RMG）与完整RSV病毒颗粒的抗体，但与完整G蛋白胞外域相比，其体外中和抗体滴度极低。我们对免疫后的小鼠采用RSV-A2株19F进行鼻腔内攻毒，结果显示免疫组动物的鼻腔与肺部病毒复制水平较未免疫对照组显著降低。RSV攻毒后病毒载量的控制效果与血清抗体结合病毒颗粒的能力呈正相关。此外，REG ΔCCD免疫组小鼠攻毒后肺部的Th2/Th1细胞因子比值极低。上述数据表明，RSV G蛋白CCD之外存在多个保护性抗原位点，这可为开发细菌表达的非糖基化G蛋白作为安全且有效的RSV疾病保护疫苗提供理论依据。