Long-Term Central and Effector SHIV-Specific Memory T Cell Responses Elicited after a Single Immunization with a Novel Lentivector DNA Vaccine

Prevention of HIV acquisition and replication requires long lasting and effective immunity. Given the state of HIV vaccine development, innovative vectors and immunization strategies are urgently needed to generate safe and efficacious HIV vaccines. Here, we developed a novel lentivirus-based DNA vector that does not integrate in the host genome and undergoes a single-cycle of replication. Viral proteins are constitutively expressed under the control of Tat-independent LTR promoter from goat lentivirus. We immunized six macaques once only with CAL-SHIV-IN− DNA using combined intramuscular and intradermal injections plus electroporation. Antigen-specific T cell responses were monitored for 47 weeks post-immunization (PI). PBMCs were assessed directly ex vivo or after 6 and 12 days of in vitro culture using antigenic and/or homeostatic proliferation. IFN-γ ELISPOT was used to measure immediate cytokine secretion from antigen specific effector cells and from memory precursors with high proliferative capacity (PHPC). The memory phenotype and functions (proliferation, cytokine expression, lytic content) of specific T cells were tested using multiparametric FACS-based assays. All immunized macaques developed lasting peripheral CD8+ and CD4+ T cell responses mainly against Gag and Nef antigens. During the primary expansion phase, immediate effector cells as well as increasing numbers of proliferating cells with limited effector functions were detected which expressed markers of effector (EM) and central (CM) memory phenotypes. These responses contracted but then reemerged later in absence of antigen boost. Strong PHPC responses comprising vaccine-specific CM and EM T cells that readily expanded and acquired immediate effector functions were detected at 40/47 weeks PI. Altogether, our study demonstrated that a single immunization with a replication-limited DNA vaccine elicited persistent vaccine-specific CM and EM CD8+ and CD4+ T cells with immediate and readily inducible effector functions, in the absence of ongoing antigen expression.预防人类免疫缺陷病毒（HIV）感染与复制需要持久且有效的免疫保护。鉴于当前HIV疫苗研发的现状，亟需创新载体与免疫策略，以开发安全且有效的HIV疫苗。本研究构建了一种新型慢病毒（Lentivirus）来源的DNA载体，该载体无法整合入宿主基因组，且仅能进行单周期复制。其病毒蛋白在山羊慢病毒来源的、不依赖Tat的长末端重复序列（LTR, Long Terminal Repeat）启动子调控下持续表达。我们联合肌内注射、皮内注射结合电穿孔技术，仅以CAL-SHIV-IN− DNA疫苗免疫6只猕猴一次。我们对免疫后（PI, Post-Immunization）47周内的抗原特异性T细胞应答进行了监测。对外周血单个核细胞（PBMC, Peripheral Blood Mononuclear Cell），我们既进行离体直接检测，也在体外培养6天、12天后，分别通过抗原刺激或稳态增殖实验开展评估。采用干扰素-γ（IFN-γ, Interferon-γ）酶联免疫斑点（ELISPOT, Enzyme-Linked Immunospot Assay）技术，我们检测了抗原特异性效应细胞以及具有高增殖能力的记忆前体细胞（PHPC, Proliferative High Precursor Cells）的即时细胞因子分泌情况。同时，基于多参数流式细胞术（FACS, Fluorescence-Activated Cell Sorting），我们检测了特异性T细胞的记忆表型与功能，包括增殖能力、细胞因子表达水平以及溶细胞活性。所有免疫猕猴均产生了持续的外周CD8+与CD4+ T细胞应答，主要针对Gag与Nef抗原。在初次扩增阶段，我们检测到了即时效应细胞，以及数量逐渐增多的、仅具备有限效应功能的增殖细胞，这类细胞表达效应记忆（EM, Effector Memory）与中枢记忆（CM, Central Memory）表型标志物。此类应答先发生收缩，随后在无抗原加强免疫的情况下于后期再次出现。在免疫后40/47周时，我们检测到了强烈的PHPC应答，这类应答由疫苗特异性的CM与EM T细胞组成，可快速增殖并获得即时效应功能。综上，本研究证实，仅接种一次复制受限的DNA疫苗，即可在无需持续抗原表达的情况下，诱导出持续存在的疫苗特异性CM与EM表型CD8+、CD4+ T细胞，这类细胞具备即时且易于诱导的效应功能。