Immunogenicity of trivalent DNA vaccine candidate encapsulated in Chitosan-TPP nanoparticles against EV-A71 and CV-A16

<b>Aim:</b> To develop a trivalent DNA vaccine candidate encapsulated in Chitosan-TPP nanoparticles against hand foot and mouth disease (HFMD) and assess its immunogenicity in mice. <b>Materials &amp; methods:</b> Trivalent plasmid carrying the VP1 and VP2 genes of EV-A71, VP1 gene of CV-A16 was encapsulated in Chitosan-TPP nanoparticles through ionic gelation. <i>In vitro</i> characterization and <i>in vivo</i> immunization studies of the CS-TPP-NPs (pIRES-VP121) were performed. <b>Results:</b> Mice administered with CS-TPP NPs (pIRES-VP121) intramuscularly were observed to have the highest IFN-γ response. Sera from mice immunized with the naked pDNA and CS-TPP-NPs (pIRES-VP121) demonstrated good viral clearance against wild-type EV-A71 and CV-A16 in RD cells. <b>Conclusion:</b> CS-TPP-NPs (pIRES-VP121) could serve as a prototype for future development of multivalent HFMD DNA vaccine candidates. Currently, there are no US-FDA approved vaccine against HFMD. China-FDA approved monovalent HFMD inactivated vaccines were unable to provide cross protection against other enterovirus serotypes. We have developed a trivalent plasmid vaccine candidate, pIRES-VP121 which encodes the VP1 and VP2 genes of EV-A71 and the VP1 gene of CV-A16. We successfully synthesized Chitosan TPP nanoparticles encapsulating DNA Encapsulation of plasmid vaccine candidate pIRES-VP121 through ionic gelation, yielding highly monodisperse nanoparticles with CS-TPP-NPs (pIRES-VP121) with &gt;70% encapsulation efficiency. Encapsulation of plasmid vaccine candidate pIRES-VP121 through ionic gelation successfully formulated sub-200 nm, spherical shaped, mildly cationic (+10 mV) and highly monodisperse CS-TPP-NPs (pIRES-VP121) with &gt;70% encapsulation efficiency. Chitosan NPs encapsulations improved T cell immunogenicity resulting in higher IFN-γ expressing CD4⁺/CD8⁺ T cells population and IFN-γ secretion compared with mice immunized with naked pIRES-VP121. Enhanced B cell neutralizing efficacy were demonstrated by mice sera (1:16 dilution) immunized with CS-TPP-NPs (pIRES-VP121) against the wild-type EV-A71 B4 and CV-A16/N132 strain enteroviruses in comparison with naked pIRES-VP121 immunization. These findings showed the potential of a trivalent DNA plasmid to serve as a prototype for future development of multivalent DNA vaccine candidate against HFMD.

研究目的：开发一种包埋于壳聚糖-三聚磷酸钠（Chitosan-TPP）纳米颗粒的三价DNA疫苗候选株，用于预防手足口病（HFMD），并评估其在小鼠体内的免疫原性。材料与方法：将携带肠道病毒71型（EV-A71）VP1、VP2基因以及柯萨奇病毒A16型（CV-A16）VP1基因的三价质粒，通过离子凝胶法包埋于壳聚糖-三聚磷酸钠纳米颗粒中。随后对CS-TPP-NPs（pIRES-VP121）开展体外（in vitro）表征与体内（in vivo）免疫接种研究。研究结果：经肌肉注射接种CS-TPP-NPs（pIRES-VP121）的小鼠，其γ干扰素（IFN-γ）应答水平最高。分别以裸质粒DNA及CS-TPP-NPs（pIRES-VP121）免疫的小鼠血清，在RD细胞中对野生型EV-A71与CV-A16均表现出良好的病毒清除效果。研究结论：CS-TPP-NPs（pIRES-VP121）可作为未来开发多价手足口病DNA疫苗候选株的原型。目前尚无美国食品药品监督管理局（US-FDA）批准的手足口病疫苗。中国国家药品监督管理局（China-FDA）批准的单价手足口病灭活疫苗，无法为其他肠道病毒血清型提供交叉保护。本研究开发了一种三价质粒疫苗候选株pIRES-VP121，其编码EV-A71的VP1、VP2基因以及CV-A16的VP1基因。本研究通过离子凝胶法成功合成了包埋该质粒疫苗候选株的壳聚糖-三聚磷酸钠纳米颗粒，所得CS-TPP-NPs（pIRES-VP121）的包封率>70%，且具有高度单分散性。通过离子凝胶法包埋质粒疫苗候选株pIRES-VP121，成功制备出粒径小于200nm、呈球形、略带正电荷（+10 mV）且高度单分散的CS-TPP-NPs（pIRES-VP121），包封率>70%。与接种裸pIRES-VP121的小鼠相比，壳聚糖纳米颗粒包埋可增强T细胞免疫原性，使表达IFN-γ的CD4+/CD8+ T细胞群体比例及IFN-γ分泌水平均得到提升。与接种裸pIRES-VP121的小鼠相比，经CS-TPP-NPs（pIRES-VP121）免疫的小鼠血清（1:16稀释度）对野生型EV-A71 B4株及CV-A16/N132株肠道病毒表现出更强的B细胞中和活性。上述研究结果表明，三价DNA质粒有望作为开发多价抗手足口病DNA疫苗候选株的原型。