单链二聚体化策略增强轮状病毒∆VP8*疫苗免疫原性

Single-chain dimerization enhances the immunogenicity of rotavirus ∆VP8* vaccine

摘要:

目的 轮状病毒(rotavirus, RV)是婴幼儿急性脱水性胃肠炎的主要病原体，目前尚无特效治疗药物，预防性疫苗接种是控制感染的有效手段之一。本研究以RV受体结合域viral protein 8^* (VP8^*)为靶点，选取其功能区域片段?VP8^* (第65-223位氨基酸)，构建单链二聚体?VP8^*-sc-dimer。采用pET-30a(+)载体在原核系统中表达并纯化该重组蛋白，评估其免疫原性和诱导中和抗体的能力，为开发安全有效的RV亚单位疫苗提供科学依据。方法 通过基因合成获得?VP8^*-sc-dimer序列，运用同源重组技术将其导入pET-30a(+)原核表达载体。将纯化的重组蛋白与佐剂AddaVax混合后，肌内注射6-7周龄BALB/c小鼠。采用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测血清中?VP8^*特异性抗体滴度，通过病毒中和试验评估免疫血清的中和活性。结果 成功表达并纯化了?VP8^*-sc-dimer重组蛋白，纯度约为90%；ELISA结果显示，?VP8^*组和?VP8^*-sc-dimer组接种后均诱导产生了?VP8^*特异性抗体，且?VP8^*-sc-dimer组抗体滴度显著高于?VP8^*组。病毒中和试验表明，2组免疫血清均可中和RV Wa株，其中?VP8^*-sc-dimer组的中和抗体滴度显著更高。结论 ?VP8^*-sc-dimer亚单位疫苗可有效刺激机体产生针对RV Wa株的高水平抗体，且免疫应答显著优于?VP8^*。具有良好免疫原性的?VP8^*单链二聚体是开发新型人类RV疫苗的重要候选抗原，具有应用前景。

Abstract:

Objective Rotavirus (RV) is a major pathogen causing acute dehydrating gastroenteritis in infants and young children. Currently, no specific therapeutic drugs are available, making preventive vaccination the most effective strategy for controlling RV infection. We targeted the RV receptor-binding domain viral protein 8^* (VP8^*) and selected its functional region ΔVP8^* (amino acids 65-223) to construct a single-chain dimer ΔVP8^*-sc-dimer. We expressed and purified this recombinant protein in a prokaryotic system using the pET-30a(+) vector and evaluated its immunogenicity and neutralizing antibody induction capacity to provide scientific evidence for developing safe and effective RV subunit vaccines.Methods The ΔVP8^*-sc-dimer sequence was synthesized and cloned into the prokaryotic expression vector pET-30a(+) via homologous recombination. The purified recombinant protein was formulated with AddaVax adjuvant and administered to 6 to 7-week-old BALB/c mice via intramuscular injection. ΔVP8^*-specific IgG antibody titers in sera were determined by enzyme-linked immunosorbent assay (ELISA), and neutralization activity of immune sera was assessed through virus neutralization assays.Results The recombinant protein ΔVP8^*-sc-dimer was successfully expressed with 90% purity. ELISA results showed that both ΔVP8^* and ΔVP8^*-sc-dimer induced specific anti-ΔVP8^* IgG antibodies following immunization, with the ΔVP8^*-sc-dimer group exhibiting significantly higher antibody titers. Virus neutralization assays revealed that immune sera from both groups neutralized the RV Wa strain, with the ΔVP8^*-sc-dimer group showing significantly superior neutralizing antibody titers.Conclusion The ΔVP8^*-sc-dimer subunit vaccine effectively stimulates high-level antibody production against RV Wa strain, demonstrating significantly enhanced immune responses compared with ΔVP8^*. With its excellent immunogenicity, ΔVP8^*-sc-dimer represents a promising candidate antigen for developing novel RV vaccines with substantial clinical application potential.