Slifka M K, Pagarigan R, Mena I, Feuer R, Whitton J L
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Virol. 2001 Mar;75(5):2377-87. doi: 10.1128/JVI.75.5.2377-2387.2001.
Coxsackievirus B3 (CVB3) is a common human pathogen that has been associated with serious diseases including myocarditis and pancreatitis. To better understand the effect of cytotoxic T-lymphocyte (CTL) responses in controlling CVB3 infection, we have inserted well-characterized CTL epitopes into the CVB3 genome. Constructs were made by placing the epitope of interest upstream of the open reading frame encoding the CVB3 polyprotein, separated by a poly-glycine linker and an artificial 3Cpro/3CDpro cleavage site. This strategy results in the foreign protein being translated at the amino- terminus of the viral polyprotein, from which it is cleaved prior to viral assembly. In this study, we cloned major histocompatibility complex class I-restricted CTL epitopes from lymphocytic choriomeningitis virus (LCMV) into recombinant CVB3 (rCVB3). In vitro, rCVB3 growth kinetics showed a 1- to 2-h lag period before exponential growth was initiated, and peak titers were approximately 1 log unit lower than for wild-type virus. rCVB3 replicated to high titers in vivo and caused severe pancreatitis but minimal myocarditis. Despite the high virus titers, rCVB3 infection of naive mice failed to induce a strong CD8+ T-cell response to the encoded epitope; this has implications for the proposed role of "cross-priming" during virus infection and for the utility of recombinant picornaviruses as vaccine vectors. In contrast, rCVB3 infection of LCMV-immune mice resulted in direct ex vivo cytotoxic activity against target cells coated with the epitope peptide, demonstrating that the rCVB3-encoded LCMV-specific epitope was expressed and presented in vivo. The preexisting CD8+ memory T cells could limit rCVB replication; compared to naive mice, infection of LCMV-immune mice with rCVB3 resulted in approximately 50-fold-lower virus titers in the heart and approximately 6-fold-lower virus titers in the pancreas. Although the inserted CTL epitope was retained by rCVB3 through several passages in tissue culture, it was lost in an organ-specific manner in vivo; a substantial proportion of viruses from the pancreas retained the insert, compared to only 0 to 1.8% of myocardial viruses. Together, these results show that expression of heterologous viral proteins by recombinant CVB3 provides a useful model for determining the mechanisms underlying the immune response to this viral pathogen.
柯萨奇病毒B3（CVB3）是一种常见的人类病原体，与包括心肌炎和胰腺炎在内的严重疾病有关。为了更好地了解细胞毒性T淋巴细胞（CTL）反应在控制CVB3感染中的作用，我们已将特征明确的CTL表位插入CVB3基因组。构建体是通过将感兴趣的表位置于编码CVB3多聚蛋白的开放阅读框上游，并由一个聚甘氨酸接头和一个人工3C蛋白酶/3CD蛋白酶切割位点隔开而制成的。这种策略导致外源蛋白在病毒多聚蛋白的氨基末端翻译，在病毒组装之前从该多聚蛋白上切割下来。在本研究中，我们将来自淋巴细胞性脉络丛脑膜炎病毒（LCMV）的主要组织相容性复合体I类限制性CTL表位克隆到重组CVB3（rCVB3）中。在体外，rCVB3的生长动力学显示在开始指数生长之前有1至2小时的延迟期，峰值滴度比野生型病毒低约1个对数单位。rCVB3在体内复制到高滴度并引起严重胰腺炎，但心肌炎轻微。尽管病毒滴度很高，但初次感染的小鼠感染rCVB3未能诱导对编码表位产生强烈的CD8 + T细胞反应；这对病毒感染期间“交叉启动”的假定作用以及重组微小核糖核酸病毒作为疫苗载体的效用具有影响。相比之下，LCMV免疫小鼠感染rCVB3导致对涂有表位肽的靶细胞具有直接的体外细胞毒性活性，表明rCVB3编码的LCMV特异性表位在体内表达并呈递。预先存在的CD8 +记忆T细胞可以限制rCVB的复制；与初次感染的小鼠相比，LCMV免疫小鼠感染rCVB3导致心脏中的病毒滴度降低约50倍，胰腺中的病毒滴度降低约6倍。尽管插入的CTL表位通过在组织培养中的几次传代被rCVB3保留，但它在体内以器官特异性方式丢失；与仅0至1.8％的心肌病毒相比，来自胰腺的大部分病毒保留了插入片段。总之，这些结果表明重组CVB3表达异源病毒蛋白为确定针对这种病毒病原体的免疫反应机制提供了一个有用的模型。
