Couillin I, Letourneur F, Lefèbvre P, Guillet J G, Martinon F
Laboratoire d'Immunologie des Pathologies Infectieuses et Tumorales, INSERM U445, Paris, France.
Virology. 2001 Jan 5;279(1):136-45. doi: 10.1006/viro.2000.0705.
CD8(+) T lymphocytes play a key role in controlling viremia during primary human immunodeficiency virus-1 and in maintaining disease-free infection. It has recently been shown that DNA immunization of rhesus monkeys can elicit strong, long-lived antigen-specific cytotoxic T lymphocyte (CTL) responses. In previous work, it was shown that macaque CTL responses to lipopeptide vaccination were directed against a limited number of epitopes. In the present study, we used the DNA immunization approach to enlarge T cell responses to several epitopes and to multiple isolates. We immunized macaques with a mixture of six plasmids reflecting the variability of Nef epitopic regions in the simian immunodeficiency virus (SIV) mac251 primary isolate. The Nef genes from viruses included in the SIVmac251 primary isolate were sequenced and the six selected sequences were individually subcloned into the pCI vector, under cytomegalovirus enhancer/promoter control, and injected into macaques. We show that DNA immunization with Nef sequences induced interferon-gamma (IFN-gamma) secreting cell responses directed against several regions of Nef. Reacting T cell lines were expanded in vitro and multispecific CTL responses mapping the 96-138 Nef region were analyzed. Several peptides recognized by CTL were identified and studies using peptides reflecting the variability of Nef indicated that all of the Nef variants were recognized in the 96-138 region. Moreover, CTL responses were directed against an immunodominant epitope located in a functional region within the Nef protein that is essential for viral replication. This work shows that our approach of DNA immunization with several sequences induced multispecific T cell responses recognizing variants included in the SIVmac251 primary isolate.
CD8(+) T淋巴细胞在控制原发性人类免疫缺陷病毒1型感染期间的病毒血症以及维持无病感染方面发挥着关键作用。最近的研究表明，恒河猴的DNA免疫能够引发强烈且持久的抗原特异性细胞毒性T淋巴细胞（CTL）反应。在先前的研究中，已表明猕猴对脂肽疫苗接种的CTL反应针对的是有限数量的表位。在本研究中，我们采用DNA免疫方法来扩大T细胞对多个表位以及多种病毒分离株的反应。我们用六种质粒的混合物免疫猕猴，这些质粒反映了猿猴免疫缺陷病毒（SIV）mac251原发性分离株中Nef表位区域的变异性。对SIVmac251原发性分离株中所含病毒的Nef基因进行测序，并将六个选定的序列分别亚克隆到受巨细胞病毒增强子/启动子控制的pCI载体中，然后注入猕猴体内。我们发现，用Nef序列进行DNA免疫可诱导针对Nef多个区域的分泌γ干扰素（IFN-γ）的细胞反应。体外扩增反应性T细胞系，并分析定位96-138 Nef区域的多特异性CTL反应。鉴定出了几种CTL识别的肽段，使用反映Nef变异性的肽段进行的研究表明，所有Nef变体在96-138区域都能被识别。此外，CTL反应针对的是位于Nef蛋白功能区域内对病毒复制至关重要的一个免疫显性表位。这项工作表明，我们用多个序列进行DNA免疫的方法诱导了识别SIVmac251原发性分离株中所含变体的多特异性T细胞反应。
