以Ⅱ型单纯疱疹病毒gD为免疫原的circRNA疫苗制备及药效分析

Preparation and efficacy of a circRNA vaccine with herpes simplex virus type II gD as immunogen

摘要:

为研究以Ⅱ型单纯疱疹病毒gD胞外域(ectodomain)为免疫原的circRNA疫苗诱导BALB/c小鼠出现的特异性免疫反应，并进行免疫研究评价，为单纯疱疹病毒mRNA疫苗的研发提供实验依据和技术参考，本研究通过PCR技术和同源重组技术，从pT7AMP-gD Ectodomain质粒中获得gD基因，将gD基因克隆至pUC57载体，获得重组pUC57-circ-gD质粒并进行测序鉴定。进行PCR获得后续实验模板DNA后，以单纯疱疹病毒囊膜糖蛋白gD Ectodomain为免疫原，并同时将模板DNA进行体外转录(in vitro transcription,IVT)及环化，将得到的pUC57-circ-gD mRNA进行RNase R酶酶切验证、反转录聚合酶链反应(reverse transcription polymerase chain reaction,RT-PCR)成环验证，以确定是否成环；转染293T细胞，72 h后收集细胞上清，进行蛋白免疫印迹法(Western blotting,WB)以鉴定蛋白表达；采用脂质纳米颗粒(lipid nanoparticles,LNP)对合成的mRNA以微流控包封法进行包封；包封后，将其应用于模型动物BALB/c小鼠中实验，在第21、35天于眼底静脉丛采血，第49天麻醉后摘除眼球采血，后续进行酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)抗体检测，并对病毒中和抗体进行测定，在第49天取脾脏，后续通过固相酶联免疫斑点技术(enzyme-linked immunospot,ELISpot)检测细胞因子IFN-γ的分泌情况。结果表明成功构建了重组质粒，测序结果正确；RNase R酶切验证后证实了环状RNA的存在，mRNA转染293T细胞后Western blotting验证有清晰的特异性条带；对疫苗进行质量检测，通过尺寸排阻高效液相色谱法(size exclusion chromatography-high performance liquid chromatography,SEC-HPLC)检测到疫苗的纯度约为90%，测量mRNA-LNP的粒径为82.76 nm，包封率达到98%左右；小鼠进行3次免疫后，在观察期内小鼠体重的增加和良好的存活率证明了该疫苗的安全性；在免疫后的小鼠血清中，IgG抗体滴度增加；在脾脏细胞中，检测到分泌特异性IFN-γ的T细胞数量升高。后续ELISA、中和抗体检测、ELISpot实验及攻毒实验说明pUC57-circ-gD mRNA免疫原性及持久性良好，并且可以抵御病毒的攻击。本研究为circRNA疫苗相关研究提供了参考。

Abstract:

This study investigated the specific immune response of BALB/c mice that was induced by a circular RNA (circRNA) vaccine expressing the herpes simplex virus type II (HSV-2) glycoprotein D (gD). The aim was to evaluate the immunological potential of this vaccine and lay a foundation for developing an mRNA vaccine against HSV-2. PCR and homologous recombination were employed to integrate the gD gene obtained from the pT7AMP-gD ectodomain plasmid into pUC57 to generate the recombinant plasmid pUC57-circ-gD, which was then sequenced and characterized. In vitro transcription and cyclization were performed on the template DNA to generate pUC57-circ-gD mRNA. To validate the formation of circular RNA, we cleaved the pUC57-circ-gD mRNA with RNase R and employed RT-PCR to validate the cyclization. The pUC57-circ-gD mRNA was then transfected into 293T cells. After 72 h, the cell supernatant was collected, and Western blotting was employed to measure the protein level of gD. Subsequently, the mRNA was encapsulated in lipid nanoparticles (LNPs) by microfluidic encapsulation. BALB/c mice were administrated with the encapsulated mRNA, and blood was collected from the fundus venous plexus after 21 and 35 days, and from the enucleated eyeballs after 49 days. Enzyme-linked immunosorbent assay was employed to measure the titers of antibodies, including virus-neutralizing antibodies. After 49 days, spleens were harvested and assessed for secretion of interferon-gamma (IFN-γ) by solid-phase enzyme-linked immunospot. The results showed successful construction and sequencing of the recombinant plasmid. RNase R digestion confirmed the presence of circular RNAs. Western blotting of the 293T cells transfected with the mRNA showed clear specific bands. The quality of the vaccine was tested by size exclusion chromatography-high performance liquid chromatography, which showed that the purity of the vaccine was about 90%. The mRNA-LNP showcased the particle size of 82.76 nm and an encapsulation rate of approximately 98%. Following three-dose vaccination, all immunized mice exhibited steady weight gain with 100% survival rate throughout the 28-day observation period, indicating no significant acute toxicity associated with the vaccine formulation. The immunized mice showed dose-dependent increases in serum IgG antibody titer and IFN-γ secretion by splenocytes and they were resistant to virus attacks. These findings indicate good immunogenicity and persistence of the pUC57-circ-gD mRNA vaccine, providing a reference for further studies on circRNA vaccines.