目的 对比呼吸道合胞病毒(RSV)融合前F蛋白(PreF)和融合后F蛋白(PostF)的免疫原性差异。 方法 通过SDS-PAGE及Western blot鉴定PreF和PostF的表达，使用Octet仪器检测F蛋白与特异性抗体的亲和力，用PreF和PostF免疫小鼠，检测免疫血清的结合抗体与中和抗体差异。 结果 PreF经改造后以稳定的三聚体形式存在，与单抗D25、8897、AM14、Palivizumab以及Motavizumab均具有很高的亲和力，但PostF缺乏表位Ø构象，并呈现单体构象，与D25、8897、AM14并无明显结合。PreF和PostF免疫小鼠的研究发现，AS01佐剂比铝佐剂更能诱导出高滴度结合抗体和针对RSV Long株的中和抗体。PreF与PostF免疫后诱导的结合抗体与PreF的结合能力相当，PostF免疫后诱导的结合抗体与PostF的结合水平显著高于PreF。 结论 PreF拥有更多的抗体结合表位，改造后稳定的PreF三聚体重组蛋白能够诱导更强的中和抗体。同时，AS01佐剂免疫效果优于铝佐剂。因此，基于PreF稳定的三聚体结构改造及佐剂的研发对于RSV疫苗的发展至关重要。 Objective To compare the immunogenicity of the prefusion (PreF) and postfusion (PostF) conformations of the respiratory syncytial virus (RSV) F protein. Methods The expression of PreF and PostF recombinant proteins was analyzed by SDS-PAGE and Western blot. The binding affinity between F protein and its specific antibodies was detected by Octet. The binding antibodies and neutralizing antibodies in immune serum were detected after immunizing mice with PreF or PostF recombinant protein. Results PreF protein was stable in the form of a trimer after modification with higher binding affinity with monoclonal antibodies such as D25, 8897, AM14, Palivizumab and Motavizumab. PostF protein lacked the antigenic site Ø and showed a monomer conformation. Besides, it was unable to bind to D25, 8897 and AM14 antibodies. Animal experiments showed that AS01 adjuvant was better than aluminum adjuvant in inducing binding antibodies and neutralizing antibodies against RSV Long strains. The binding antibodies induced by PreF and PostF recombinant proteins had similar binding ability to PreF protein, while the binding antibodies induced by PostF recombinant protein showed stronger binding ability to PostF than to PreF. Conclusions PreF has more epitopes and the trimer form of PreF recombinant protein after modification is more stable and can induce stronger neutralizing antibodies. Moreover, the immunopotentiating effect of AS01 adjuvant is better than that of aluminum adjuvant. Therefore, stabilization-based trimer structure modification of PreF and the development of adjuvants are crucial for the development of RSV vaccines.
Immunogenicity of PreF and PostF recombinant protein vaccines against respiratory syncytial virus
Objective To compare the immunogenicity of the prefusion (PreF) and postfusion (PostF) conformations of the respiratory syncytial virus (RSV) F protein. Methods The expression of PreF and PostF recombinant proteins was analyzed by SDS-PAGE and Western blot. The binding affinity between F protein and its specific antibodies was detected by Octet. The binding antibodies and neutralizing antibodies in immune serum were detected after immunizing mice with PreF or PostF recombinant protein. Results PreF protein was stable in the form of a trimer after modification with higher binding affinity with monoclonal antibodies such as D25, 8897, AM14, Palivizumab and Motavizumab. PostF protein lacked the antigenic site Ø and showed a monomer conformation. Besides, it was unable to bind to D25, 8897 and AM14 antibodies. Animal experiments showed that AS01 adjuvant was better than aluminum adjuvant in inducing binding antibodies and neutralizing antibodies against RSV Long strains. The binding antibodies induced by PreF and PostF recombinant proteins had similar binding ability to PreF protein, while the binding antibodies induced by PostF recombinant protein showed stronger binding ability to PostF than to PreF. Conclusions PreF has more epitopes and the trimer form of PreF recombinant protein after modification is more stable and can induce stronger neutralizing antibodies. Moreover, the immunopotentiating effect of AS01 adjuvant is better than that of aluminum adjuvant. Therefore, stabilization-based trimer structure modification of PreF and the development of adjuvants are crucial for the development of RSV vaccines.
Respiratory syncytial virusRecombinant F proteinPrefusion conformationAdjuvantsImmunogenicity evaluation
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