Project 3: Determination of the minimal MHC-E-restricted SIV epitope targeting required for RhCMV/SIV vaccine-mediated SIV replication arrest efficacy

项目3--项目总结 恒河猴68-1株巨细胞病毒疫苗载体表达猴的免疫应答 在59%的人中，免疫缺陷病毒(SIV)插入物(RhCMV/SIV)严格控制SIV(通过复制停止) 疫苗接种者，随后随着时间的推移病毒清除。这种疫苗介导复制抑制的能力已经 与其诱导SIV特异性CD8+T细胞识别病毒多肽的能力密切相关 功能上非多态的主要组织相容性复合体(MHC)-E而不是经典的高度多态 MHC-Ia。这些MHC-E限制性CD8+T细胞反应在大规模病毒感染之前拦截原始SIV感染 导致突变逃逸的复制和序列多样化，因此它们可能只需要有效的 整体的SIV识别，而不一定是表位的广度。我们小组的初步工作发现，MHC- E限制性TCR和T细胞克隆型与经典MHC-Ia限制性TCR和T细胞克隆型有根本的不同 CD8+T细胞反应。而传统的MHC-Ia限制性表位靶向反应是由 独立的TCR，MHC-E限制的SIV特异性反应(即使针对许多SIV表位) 主要由少数(&lt；10)相对低亲和度和高亲和度的独立TCR组成 交叉反应-每个TCR识别一个或多个称为超表位的通用表位(表位 被所有68-1 RhCMV/SIV疫苗接种的RMS识别)和数量可变的各种非通用表位。 在这个项目中，我们试图定义最小的MHC-E限制性CD8+T细胞TCR识别单元，能够 调解SIV复制停滞型疗效，询问疗效是否需要承认 一个或多个超群，以及这种有效识别是否需要一个或多个TCR克隆型 (有效地询问多个SIV表位的交叉反应TCR识别是否是内在要求 用于有效的整体SIV感染细胞识别，从而用于疗效)。此外，鉴于缺乏MHC-E 多态，我们问：开发一种通用的MHC-E限制性超抗原表位靶向HIV疫苗是否 可能--一种对所有接种者都有类似特异性并具有足够广泛靶向性的疫苗，以确保疗效- 充分认识到全球艾滋病毒序列的可变性。这些问题将通过以下方式得到回答 以下具体目标：S.A.1-设计和验证miR-126受限(仅限MHC-E)68-1的SIV插入件 将(排他地)诱导SIV MHC-E限制性SIV特异性CD8+T细胞应答的RhCMV载体 1、2和10个SIV表型；S.A.2-比较免疫原性(包括反应特异性、TCR克隆型 单MHC-E、双MHC-E和10 MHC-E的层级结构和TCR交叉反应)和复制停滞型疗效 以及S.A.3-开发体外工程的自体T细胞，表达有效的MHC- 在SIV挑战时用于体内输注的E-限制性SIV超抗原表位特异性TCRs 表达保护性MHC-E限制性TCRs的CD8+T细胞在缺失时可介导复制抑制效应 接种人巨细胞病毒/SIV疫苗。

PROJECT 3 - PROJECT SUMMARY Immune responses elicited by strain 68-1 Rhesus Cytomegalovirus vaccine vectors expressing simian immunodeficiency virus (SIV) inserts (RhCMV/SIV) stringently control SIV (via “replication arrest”) in 59% of vaccinees, with subsequent viral clearance over time. The ability of this vaccine to mediate replication arrest has been strongly linked to its ability to elicit SIV-specific CD8+ T cells that recognize viral peptides in the context of functionally non-polymorphic major histocompatibility complex (MHC)-E rather than classical highly polymorphic MHC-Ia. These MHC-E-restricted CD8+ T cell responses intercept primary SIV infection before the massive viral replication and sequence diversification that leads to mutational escape, and thus they may only require effective overall SIV recognition and not necessarily epitope breadth. Preliminary work by our group has found that MHC- E-restricted TCRs and T cell clonotypes are fundamentally different from those of classical MHC-Ia-restricted CD8+ T cell responses. Whereas conventional MHC-Ia-restricted epitope-targeted responses are mediated by independent TCRs, MHC-E-restricted SIV-specific responses (even when directed at numerous SIV epitopes) are largely comprised of only a handful (<10) of independent TCRs that are relatively low avidity and highly cross-reactive – with each TCR recognizing one or more universal epitopes called supertopes (epitopes that are recognized by all 68-1 RhCMV/SIV vaccinated RMs) and a variable number of diverse non-universal epitopes. In this project we seek to define the minimal MHC-E-restricted CD8+ T cell TCR recognition unit capable of mediating SIV replication arrest-type efficacy, asking the questions of whether efficacy requires recognition of one or multiple supertopes, and whether this efficacious recognition requires one or multiple TCR clonotypes (effectively asking whether cross-reactive TCR recognition of multiple SIV epitopes is an intrinsic requirement for effective overall SIV-infected cell recognition and thus for efficacy). Also, given the lack of MHC-E polymorphism, we ask whether development of a universal MHC-E-restricted supertope-targeted HIV vaccine is possible – a vaccine that has similar specificity in all vaccinees and is broadly targeted enough to ensure efficacy- sufficient recognition across global HIV sequence variability. These questions will be answered by addressing the following Specific Aims: S.A.1 - to design and validate SIV inserts for miR-126-restricted (MHC-E-only) 68-1 RhCMV vectors that will (exclusively) elicit SIV MHC-E-restricted SIV-specific CD8+ T cell responses limited to 1, 2, and 10 SIV supertopes; S.A.2 - to compare immunogenicity (including response specificity, TCR clonotypic hierarchies, and TCR cross-reactivity) and replication arrest-type efficacy of single, dual and 10 MHC-E supertope-only vectors; and S.A.3 - to develop ex vivo engineered autologous T cells expressing validated MHC- E-restricted SIV supertope-specific TCRs for in vivo infusion at the time of SIV challenge to determine whether CD8+ T cells expressing protective MHC-E-restricted TCRs can mediate replication arrest efficacy in the absence of RhCMV/SIV vaccination.