Starostina Ekaterina V, Sharabrin Sergei V, Antropov Denis N, Stepanov Grigory A, Shevelev Georgiy Yu, Lemza Anna E, Rudometov Andrey P, Borgoyakova Mariya B, Rudometova Nadezhda B, Marchenko Vasiliy Yu, Danilchenko Natalia V, Chikaev Anton N, Bazhan Sergei I, Ilyichev Alexander A, Karpenko Larisa I
State Research Center of Virology and Biotechnology "Vector", Koltsovo, 630559 Novosibirsk, Russia.
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia.
Vaccines (Basel). 2021 May 3;9(5):452. doi: 10.3390/vaccines9050452.
Nucleic acid-based influenza vaccines are a promising platform that have recently and rapidly developed. We previously demonstrated the immunogenicity of DNA vaccines encoding artificial immunogens AgH1, AgH3, and AgM2, which contained conserved fragments of the hemagglutinin stem of two subtypes of influenza A-H1N1 and H3N2-and conserved protein M2. Thus, the aim of this study was to design and characterize modified mRNA obtained using the above plasmid DNA vaccines as a template. To select the most promising protocol for creating highly immunogenic mRNA vaccines, we performed a comparative analysis of mRNA modifications aimed at increasing its translational activity and decreasing toxicity. We used mRNA encoding a green fluorescent protein (GFP) as a model. Eight mRNA-GFP variants with different modifications (M0-M7) were obtained using the classic cap(1), its chemical analog ARCA (anti-reverse cap analog), pseudouridine (Ψ), N6-methyladenosine (m6A), and 5-methylcytosine (m5C) in different ratios. Modifications M2, M6, and M7, which provided the most intensive fluorescence of transfected HEK293FT cells were used for template synthesis when mRNA encoded influenza immunogens AgH1, AgH3, and AgM2. Virus specific antibodies were registered in groups of animals immunized with a mix of mRNAs encoding AgH1, AgH3, and AgM2, which contained either ARCA (with inclusions of 100% Ψ and 20% m6A (M6)) or a classic cap(1) (with 100% substitution of U with Ψ (M7)). M6 modification was the least toxic when compared with other mRNA variants. M6 and M7 RNA modifications can therefore be considered as promising protocols for designing mRNA vaccines.
基于核酸的流感疫苗是一个很有前景的平台，近年来发展迅速。我们之前证明了编码人工免疫原AgH1、AgH3和AgM2的DNA疫苗的免疫原性，这些免疫原包含甲型流感病毒H1N1和H3N2两个亚型血凝素茎的保守片段以及保守蛋白M2。因此，本研究的目的是设计并表征以上述质粒DNA疫苗为模板获得的修饰mRNA。为了选择最有前景的方案来制备高免疫原性的mRNA疫苗，我们对旨在提高其翻译活性和降低毒性的mRNA修饰进行了比较分析。我们使用编码绿色荧光蛋白（GFP）的mRNA作为模型。使用经典帽结构（1）、其化学类似物ARCA（抗反向帽类似物）、假尿苷（Ψ）、N6-甲基腺苷（m6A）和5-甲基胞嘧啶（m5C）以不同比例获得了八个具有不同修饰（M0-M7）的mRNA-GFP变体。当mRNA编码流感免疫原AgH1、AgH3和AgM2时，将在转染的HEK293FT细胞中产生最强荧光的修饰M2、M6和M7用于模板合成。在用编码AgH1、AgH3和AgM2的mRNA混合物免疫的动物组中检测到了病毒特异性抗体，这些mRNA混合物要么含有ARCA（包含100%的Ψ和20%的m6A（M6）），要么含有经典帽结构（1）（U被Ψ 100%取代（M7））。与其他mRNA变体相比，M6修饰的毒性最小。因此，M6和M7 RNA修饰可被视为设计mRNA疫苗的有前景的方案。
