模块化HBc-VLPs载体微针疫苗的制备及其特性和免疫效果分析

Preparation,characteristics and immune effect of modular HBc-VLPs carrier microneedle vaccine

目的制备模块化HBc-VLPs载体微针疫苗,并分析其特性和免疫效果。方法通过定向改造hbc的基因序列,制备HBc(A80C)单体蛋白,纯化后的单体蛋白自组装形成HBc-VLPs纳米载体。利用Sulfo-SMCC将含有-NH2的流感病毒模式抗原肽M2e锚定于HBc-VLPs表面的Cys位点,制备M2e-HBc-VLPs模式抗原。以快速溶解材料为基质,将抗原微粒混合于基质中,制备涂层微针疫苗,并进行凝胶穿刺、皮肤穿刺及疫苗释放试验,Western blot分析M2e-HBc单体的抗原性。将剂量分别为50和100μg/只的微针涂层疫苗经皮内免疫BALB/c小鼠,间接ELISA法检测小鼠血清特异性抗体效价,流式细胞术检测小鼠T淋巴细胞变化。结果 HBc(A80C)单体组装形成了粒径约为30 nm的HBc-VLPs球形载体。凝胶、皮肤穿刺试验表明,抗原能够到达表皮层,并在15 min内完成90%以上的释放。偶联M2e抗原能与相应抗体发生特异性结合,疫苗能够有效刺激小鼠产生抗原特异性IgG抗体,并在一定程度上激活机体的细胞免疫,验证了模式疫苗的有效性。结论具备定点偶联功能的HBc-VLPs载体能够搭载多种抗原模块,形成针对性的疫苗,缩短疫苗在制备、评价等方面所需时间,加快疫苗的研发流程。通过微针进行皮肤免疫,能够实现大规模的快速接种。本研究为HBc-VLPs作为模块化疫苗载体奠定了基础,可促进模块化微针疫苗的研发。

Objective To prepare modular HBc-VLPs carrier microneedle vaccine and analyze its characteristics and immune effect. Methods By directional transformation of hbc gene sequence,HBc(A80C)monomer protein was prepared,pu-rified and self-assembled to form HBc-VLP nanocarriers. The influenza virus antigen M2e peptide as model antigen was connected to Cys residue of HBc-VLPs by Sulfo-SMCC to prepare M2e-HBc-VLPs model antigen. Antigen particles were mixed in a rapid-dissolving material as a matrix to prepare a coated microneedle vaccine which was subjected to gel puncture,skin puncture and release tests. The antigenicity of M2e-HBc monomer was analyzed by Western blot. BALB/c mice were immunized with the coated microneedle vaccine by intradermal injection at dosages of 50 and 100 μg respectively,of which the serum specific antibody titer was determined by indirect ELISA,and the T lymphocyte level by flow cytometry. Results HBc(A80C) monomer was assembled to form HBc-VLPs spherical carrier at a particle size of about 30 nm. Gel and skin puncture tests showed that the antigen reached the epidermal layer,of which more than 90% was released within 15 min. The conjugated M2e antigen specifically bound to the corresponding antibody. The vaccine effectively stimulated mice to produce antigen-specific IgG antibodies and activated the cellular immunity,which validated the effectiveness of the model vaccine. Conclusion The HBc-VLPs carrier with site-directed coupling function can carry a variety of antigen modules to form a targeted vaccine,shorten the time required for vaccine preparation and evaluation,and accelerate the vaccine development process. Large-scale rapid vaccination may be performed through skin immunization with microneedles. The research results lay a foundation of application of HBc-VLPs as a modular vaccine carrier,which can promote the development of modular microneedle vaccines.