Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate
Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate
复制标题
DOI：
10.1128/jvi.00189-17
复制
发表时间：
2017-08-01
影响因子：
5.4
通讯作者：
Munir, Shirin
中科院分区：
文献类型：
--
作者：
Liang, Bo;Ngwuta, Joan O.;Munir, Shirin
呼吸道合胞病毒（Respiratory syncytial virus，RSV）是世界范围内小儿严重呼吸道疾病的主要病原体，但目前尚无有效的疫苗和抗病毒药物。先前开发了活的减毒嵌合牛/人副流感病毒3型（rB/HPIV 3）作为表达RSV融合（F）蛋白的载体，以赋予针对RSV和HPIV 3的二价保护。在既往病毒初治儿童的临床试验中，rB/HPIV 3耐受良好，但野生型RSV F的免疫原性不令人满意。我们先前用设计的二硫键（DS）修饰RSV F以增加融合前（pre-F）构象的稳定性并有效地包装在载体病毒体中。在这里，我们通过添加二硫键和空腔填充突变（DS-Cav 1）进一步稳定了前F，并且我们还修改了RSV F密码子使用以具有较低的CpG含量和较高的表达水平。在rB/HPIV 3中以三种形式评估该RSV F开放阅读框架：（i）无载体包装信号的前F，（ii）具有载体包装信号的前F，和（iii）分泌的前F胞外域三聚体。尽管被有效地表达，分泌的前F是免疫原性差。DS-Cav 1稳定前F，有或没有包装，诱导仓鼠中前F特异性抗体的更高滴度，并提高RSV中和血清抗体的质量。含有较少CpG二核苷酸的密码子优化的RSV F具有更高的F表达，在体内更有效地复制，并且更具免疫原性。DS-Cav 1 pre-F稳定化、优化的密码子使用、降低的CpG含量和载体包装的组合显著改善了载体免疫原性和针对RSV的保护效力。这提供了一种适合儿科临床评价的改进的载体RSV候选疫苗。重要信息RSV和HPIV 3是全球严重儿科呼吸道疾病的第一和第二大病毒原因。没有获得许可的疫苗或合适的抗病毒药物。我们正在开发一种表达RSV F的嵌合rB/HPIV 3载体作为二价RSV/HPIV 3疫苗，并一直在评估增加RSV F免疫原性的方法。在这项研究中，我们评估了前F构象中F的稳定性提高和密码子优化导致CpG含量降低和前F表达增加的影响。CpG含量的减少抑制了干扰素对感染的反应，促进了更高的复制和F表达的增加。我们证明，改进的前F稳定性和密码子使用的策略性操作，以及有效的前F包装到载体病毒体中，显著增加了二价RSV/HPIV 3疫苗中的F免疫原性。改善的免疫原性包括诱导高质量补体非依赖性抗体的滴度增加，具有更大的前F位点O结合和更大的抗RSV攻击保护。
Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory tract disease worldwide, but it lacks a licensed vaccine or suitable antiviral drug. A live attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) was developed previously as a vector expressing RSV fusion (F) protein to confer bivalent protection against RSV and HPIV3. In a previous clinical trial in virus-naive children, rB/HPIV3 was well tolerated but the immunogenicity of wild-type RSV F was unsatisfactory. We previously modified RSV F with a designed disulfide bond (DS) to increase stability in the prefusion (pre-F) conformation and to be efficiently packaged in the vector virion. Here, we further stabilized pre-F by adding both disulfide and cavity-filling mutations (DS-Cav1), and we also modified RSV F codon usage to have a lower CpG content and a higher level of expression. This RSV F open reading frame was evaluated in rB/HPIV3 in three forms: (i) pre-F without vector-packaging signal, (ii) pre-F with vector-packaging signal, and (iii) secreted pre-F ectodomain trimer. Despite being efficiently expressed, the secreted pre-F was poorly immunogenic. DS-Cav1 stabilized pre-F, with or without packaging, induced higher titers of pre-F specific antibodies in hamsters, and improved the quality of RSV-neutralizing serum antibodies. Codon-optimized RSV F containing fewer CpG dinucleotides had higher F expression, replicated more efficiently in vivo, and was more immunogenic. The combination of DS-Cav1 pre-F stabilization, optimized codon usage, reduced CpG content, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved vectored RSV vaccine candidate suitable for pediatric clinical evaluation.IMPORTANCE RSV and HPIV3 are the first and second leading viral causes of severe pediatric respiratory disease worldwide. Licensed vaccines or suitable antiviral drugs are not available. We are developing a chimeric rB/HPIV3 vector expressing RSV F as a bivalent RSV/HPIV3 vaccine and have been evaluating means to increase RSV F immunogenicity. In this study, we evaluated the effects of improved stabilization of F in the pre-F conformation and of codon optimization resulting in reduced CpG content and greater pre-F expression. Reduced CpG content dampened the interferon response to infection, promoting higher replication and increased F expression. We demonstrate that improved pre-F stabilization and strategic manipulation of codon usage, together with efficient pre-F packaging into vector virions, significantly increased F immunogenicity in the bivalent RSV/HPIV3 vaccine. The improved immunogenicity included induction of increased titers of high-quality complement-independent antibodies with greater pre-F site O binding and greater protection against RSV challenge.