Arrode-Brusés Géraldine, Moussa Maha, Baccard-Longere Monique, Villinger François, Chebloune Yahia
INRA, ANRS, Université Joseph Fourier, PAVAL Lab./Nanobio 2, UJF Grenoble, Grenoble, France.
Institut de Biologie et Pathologie, Centre Hospitalo-Universitaire de Grenoble, Grenoble, France.
PLoS One. 2014 Oct 22;9(10):e110883. doi: 10.1371/journal.pone.0110883. eCollection 2014.
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HIV感染和复制需要持久且有效的免疫力。鉴于HIV疫苗的研发现状，迫切需要创新的载体和免疫策略来开发安全有效的HIV疫苗。在此，我们开发了一种新型的基于慢病毒的DNA载体，该载体不会整合到宿主基因组中，且仅经历一个复制周期。病毒蛋白在山羊慢病毒的不依赖Tat的LTR启动子控制下持续表达。我们仅对6只猕猴进行了一次免疫，采用肌肉注射、皮内注射联合电穿孔的方式接种CAL-SHIV-IN-DNA。在免疫后（PI）47周监测抗原特异性T细胞反应。使用抗原性和/或稳态增殖直接在体外评估外周血单核细胞（PBMC），或在体外培养6天和12天后进行评估。使用IFN-γ ELISPOT来测量抗原特异性效应细胞和具有高增殖能力的记忆前体细胞（PHPC）的即时细胞因子分泌。使用基于多参数流式细胞术的检测方法测试特异性T细胞的记忆表型和功能（增殖、细胞因子表达、杀伤成分）。所有免疫的猕猴均产生了持久的外周CD8⁺和CD4⁺ T细胞反应，主要针对Gag和Nef抗原。在初次扩增阶段，检测到即时效应细胞以及效应功能有限的增殖细胞数量增加，这些细胞表达效应记忆（EM）和中央记忆（CM）表型的标志物。这些反应有所收缩，但随后在没有抗原增强的情况下再次出现。在免疫后40/47周检测到由疫苗特异性CM和EM T细胞组成的强烈PHPC反应，这些细胞易于扩增并获得即时效应功能。总之，我们的研究表明，在没有持续抗原表达的情况下，用复制受限的DNA疫苗进行单次免疫可引发持久的疫苗特异性CM和EM CD8⁺和CD4⁺ T细胞，并具有即时且易于诱导的效应功能。
