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 -项目概要 猴巨细胞病毒68-1株疫苗载体诱导的免疫应答 免疫缺陷病毒（SIV）插入物（RhCMV/SIV）严格控制SIV（通过“复制停滞”）在59%的 疫苗接种者，随着时间的推移，随后的病毒清除。这种疫苗介导复制停滞的能力 这与其诱导SIV特异性CD 8 + T细胞的能力密切相关，该细胞在感染的背景下识别病毒肽。 主要组织相容性复合体（MHC）-E的功能非多态性，而不是经典的高度多态性 MHC-Ia这些MHC-E限制性CD 8 + T细胞应答在大量病毒感染前阻断了原发性SIV感染。 复制和序列多样化，导致突变逃逸，因此它们可能只需要有效的 总的SIV识别，而不一定是表位宽度。我们小组的初步工作发现，MHC- E限制性TCR和T细胞克隆型与经典的MHC-Ia限制性TCR和T细胞克隆型根本不同。 CD 8 + T细胞应答。而常规的MHC-Ia限制性表位靶向应答是由 独立的TCR，MHC-E限制的SIV特异性应答（即使针对许多SIV表位） 主要由少数（<10个）独立的TCR组成，这些TCR具有相对低的亲合力和高度的亲和力。 交叉反应-每个TCR识别一个或多个称为超表位的通用表位（ 由所有68-1 RhCMV/SIV疫苗接种的RM识别）和可变数量的不同非通用表位。 在这个项目中，我们试图定义最小的MHC-E限制性CD 8 + T细胞TCR识别单位， 介导SIV复制抑制型功效，询问功效是否需要识别 一个或多个超表位，以及这种有效的识别是否需要一个或多个TCR克隆型 （有效地询问多个SIV表位的交叉反应性TCR识别是否是内在要求） 用于有效的总体SIV感染的细胞识别并因此用于功效）。另外，由于缺乏MHC-E 多态性，我们问是否开发一种通用的MHC-E限制性超表位靶向HIV疫苗， 可能的-一种疫苗，具有类似的特异性，在所有疫苗接种者和广泛的目标，足以确保疗效- 对全球HIV序列变异性的充分识别。这些问题将通过解决 S.A.1 -设计和验证miR-126限制性（仅MHC-E）68-1的SIV插入片段 RhCMV载体将（排他地）引发SIV MHC-E限制性SIV特异性CD 8 + T细胞应答，其限于 1、2和10个SIV超表位; S.A.2 -比较免疫原性（包括应答特异性、TCR克隆型 分级和TCR交叉反应性）和复制抑制型MHC-E单，双和10的功效 S.A.3 -开发表达经验证的MHC的离体工程化自体T细胞- 在SIV攻击时用于体内输注的E-限制性SIV超表位特异性TCR，以确定是否 表达保护性MHC-E-限制性TCR的CD 8 + T细胞可以在缺乏免疫抑制剂的情况下介导复制阻滞功效。 RhCMV/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.