Leveraging mRNAs sequences to express SARS-CoV-2 antigens in vivo

Chunxi Zeng; Xucheng Hou; Jingyue Yan; Chengxiang Zhang; Wenqing Li; Weiyu Zhao; Shi Du; Yizhou Dong

Given the potent activity of NASAR mRNAs and urgent demand for SARS-CoV-2 vaccines, we aim to create NASAR mRNAs encoding SARS-CoV-2 antigens. According to previous studies on SARS-CoV, its structural spike (S) and nucleocapsid (N) proteins, regarded as the dominant antigens eliciting neutralizing antibodies in SARS-CoV patients, were explored as vaccine candidates 38, 39 . The receptor binding domain (RBD) of the S protein responsible for viral entry was also utilized for vaccine development 38 . The membrane (M) protein contained epitopes 40 . Additionally, the envelope (E) protein interacting with M was indispensable for the assembly of viral particles 41 . Therefore, five NASAR mRNAs were prepared to express RBD, S, N, M, and E proteins as the vaccine candidates. Since no antibodies have been developed against these five potential antigens in SARS-CoV-2, we installed a FLAG tag to RBD andS protein , and a VSV-G tag to N, M, and E proteins in order to detect their expression. To confirm the effects of UTRs on antigen expression using NASAR mRNA vaccine, three additional mRNAs were synthesized using the coding sequence of RBD with control UTRs, CYBA, MOD2, and NASAR. After the delivery of these three mRNAs into 293T cells using our previously developed lipid like-nanoparticles, TT3 42 , RBD expression was analyzed by an ELISA. NASAR enabled antigen expression 3-fold of CYBA and 1.6-fold of MOD2 (Fig. 5a) , consistent with the luciferase expression results in Fig. 4c . Then, NASAR mRNAs encoding all five SARS-CoV-2 antigens were delivered to 293T cells by TT3 separately. We observed the obvious expression of these antigens through immunostaining (Fig. 5b, Supplementary Fig. 4) .