Novel Chikungunya vaccine with rearranged genome

摘要 基孔肯雅热病毒（Chikungunya fever virus，CHIKV）是一种蚊媒甲病毒， 感染和流行病在亚洲，非洲和最近，在美洲。CHIKV风险增加 由于气候和生态变化以及国际旅行，目前，没有批准 疫苗以前的实验方法包括美国陆军开发的IND疫苗181/25， 在E2蛋白中含有两个减弱突变。181/25疫苗免疫原性高， II期临床试验;然而，不良反应证明需要改善安全性。梅迪根有 开发了一种DNA启动的减毒活疫苗，称为iDNA®，其中， 181/25 IND疫苗病毒从质粒上的CMV启动子转录。质粒注射 肌肉注射释放减毒活疫苗病毒。这种方法具有DNA的优点 疫苗，包括由遗传稳定的DNA质粒编码的疫苗病毒，和高效力 减毒活疫苗 为了满足提高181/25疫苗安全性的需要，我们提出了基因组重排， 一个额外的减毒突变这种基因组重排（RA 181/25）不会改变免疫原性 表位，但对逆转具有抗性，因为逆转需要多次独立的突变。在 具体目的1，我们制备了重排（RA 181/25）CHIKV iDNA疫苗，并对其进行了体外空斑评价 表型、生长曲线和遗传稳定性（通过下一代测序（NGS）），与非 重排的iDNA和181/25 IND疫苗病毒。在特异性目的2中，在以下中评价RA 181/25 CHIKV iDNA： 两种小鼠模型（近交系C57 BL/6和远交系CD-1）与非重排CHIKV iDNA 181/25 IND疫苗病毒我们建议使用电穿孔、微针和脂质体 我们还将评估iDNA疫苗的安全性、免疫原性（中和）、 抗体和细胞介导的应答），以及在两种动物中针对CHIKV攻击的保护功效 模型RA 181/25 CHIKV iDNA安全性优势的成功概念验证 疫苗将促进一种新的单剂量疫苗，该疫苗具有许多优点，可保护处于危险中的人群 用于CHIKV感染以及用于快速控制CHIKV爆发。

ABSTRACT Chikungunya fever virus (CHIKV) is a mosquito-borne alphavirus that causes wide-spread human infections and epidemics in Asia, Africa and recently, in the Americas. There is an increased risk of CHIKV pandemic due to climate and ecological change and international travel. Currently, there is no approved vaccine. Previous experimental approaches include the US Army-developed IND vaccine, 181/25, that contains two attenuating mutations in the E2 protein. The 181/25 vaccine showed high immunogenicity in Phase II clinical trial; however, adverse reactions justify the need for safety improvement. Medigen has developed a DNA-launched, live-attenuated vaccine, termed iDNA®, in which the full-length genome of the 181/25 IND vaccine virus is transcribed from a CMV promoter on a plasmid. Injection of the plasmid intramuscularly launches the live-attenuated vaccine virus. This approach has the advantages of DNA vaccines, including the encoding of the vaccine virus by a genetically stable DNA plasmid, and the high efficacy of live-attenuated vaccines. To address the need for improved safety of the 181/25 vaccine, we propose a genomic rearrangement as an additional attenuating mutation. This genomic rearrangement (RA181/25) does not change immunogenic epitopes but is resistant to reversion since multiple, independent mutations would be needed for reversion. In Specific Aim 1, we prepare the rearranged (RA181/25) CHIKV iDNA vaccine and evaluate it in vitro for plaque phenotype, growth curve, and genetic stability by next generation sequencing (NGS), as compared to non- rearranged iDNA and the 181/25 IND vaccine virus. In Specific Aim 2, RA181/25 CHIKV iDNA is evaluated in two mouse models (inbred C57BL/6 and outbred CD-1) as compared to the non-rearranged CHIKV iDNA and the 181/25 IND vaccine virus. We propose to use electroporation, microneedles, and liposomal formulations to optimize the iDNA vaccination, and we will evaluate safety, immunogenicity (neutralizing antibody and cell-mediated responses), and protective efficacy against CHIKV challenge in both animal models. The successful proof-of-concept demonstration of a safety advantage of the RA181/25 CHIKV iDNA vaccine would advance a novel single-dose vaccine with many advantages for protecting populations at risk for CHIKV infection, as well as for rapidly controlling CHIKV outbreaks.