Organism-level analysis of vaccination reveals networks of protection across tissues

Whole organs or CD8+ T cell populations sorted by FACS from tissues were isolated at various time points following vaccination with Vaccinia virus strain Modified Vaccinia Ankara (MVA; subcutaneously) and/or infection with strain Western Reserve (WR; intranasally). Total RNA was extracted from whole organs or sorted cell populations, RNA-seq libraries were prepared with a custom protocol, and sequenced at an average depth of ~5M/sample. We compared three types of immune responses in vivo: (1) vaccinating (MVA only; 10^7 PFUs subcutaneously), (2) lethal (WR only; 10^7 PFUs intranasally), and (3) protective (MVA followed by WR challenge; same doses and routes as in 1 and 2, respectively). Overall, 6 datasets were generated from the following experiments and samples: (i) Whole-tissue samples from MVA, WR, MVA+WR cohorts with WR challenge at day 7, 21 (Exp. 1), or 80 (Exp. 2) in wild-type mice; (ii) Whole-tissue samples from MVA+WR at day 21 in wild-type versus knockout animals: Tcra-/- (Exp. 3), and Ifng-/-, Il22-/-, and Csf2-/- (Exp. 5); (iii) Sorted CD8+ T cell populations profiled from tissues of MVA-vaccinated (day 21) and control mice with (Exp. 6) or without (Exp. 4) intravascular staining (anti-CD45-PE antibody) to distinguish parenchyma-associated from vasculature-associated cells.

本研究在以痘苗病毒减毒株Ankara（Modified Vaccinia Ankara, MVA；皮下接种）以及/或Western Reserve毒株（WR；鼻内感染）免疫接种后的不同时间点，从组织中分离完整器官，或通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）分选得到CD8+ T细胞群。从完整器官或分选得到的细胞群中提取总RNA，采用自定义实验方案制备RNA测序（RNA-seq）文库，并以约500万条reads/样本的平均测序深度完成测序。本研究比较了三类体内免疫应答：(1) 单纯免疫组（仅接种MVA；10^7噬斑形成单位（Plaque Forming Unit, PFU）皮下注射）；(2) 致死感染组（仅感染WR；10^7 PFU鼻内接种）；(3) 保护免疫组（先接种MVA，再以WR攻毒；剂量与接种途径分别与(1)(2)一致）。最终从以下实验与样本中生成了共计6套数据集：(i) 野生型小鼠中，分别在免疫后第7、21天（实验1）及第80天（实验2）的MVA单独接种、WR单独感染及MVA+WR攻毒队列的全组织样本；(ii) 免疫后第21天的MVA+WR队列全组织样本，分别取自野生型小鼠与基因敲除小鼠：包括Tcra-/-小鼠（实验3），以及Ifng-/-、Il22-/-与Csf2-/-小鼠（实验5）；(iii) 经MVA免疫21天的小鼠及对照小鼠的组织中分选得到的CD8+ T细胞群，其中部分小鼠接受了血管内染色（抗CD45-PE抗体）以区分实质组织相关细胞与脉管系统相关细胞（实验6），其余未进行该染色操作（实验4）。