Human cytotoxic T-cell responses against Epstein-Barr virus nuclear antigens demonstrated by using recombinant vaccinia viruses.

The potentially pathogenic effects of infection with Epstein-Barr virus (EBV), a B-lymphotropic agent with cell growth-transforming potential, are contained in healthy virus carriers by virus-specific cytotoxic T-lymphocyte (CTL) surveillance. The target antigens against which such CTL responses are directed are yet undefined, but the antigens probably derived from one or more of the EBV "latent" proteins constitutively expressed in virus-transformed B cells. We have analyzed target specificity of CTL responses from two EBV-immune donors that are preferentially reactive against autologous cells transformed with type A but not with type B virus isolates. Coding sequences for four EBV latent proteins with allelic polymorphism between A and B virus types--namely, the EBV nuclear antigens (EBNAs) EBNA 2, EBNA 3a, EBNA 3c, and EBNA leader protein--have been introduced into vaccinia virus vectors under control of vaccinia promoter P7.5 and used to express relevant EBNA proteins in appropriate target cells. Thus the CTL response from one donor has been mapped to type A EBNA 2 protein and from a second donor to type A EBNA 3a protein. Thereafter, a series of recombinant vaccinia viruses were constructed that carried specific internal deletions within the EBNA 2 type A coding sequence; by using these vectors, the above EBNA 2 type A-specific CTL response was shown to be directed against an epitope within a 100-amino acid fragment near the N terminus of the protein. This work clearly shows human CTL recognition of virus-coded nuclear antigens in the EBV system; moreover, it establishes an experimental approach that can be extended to all EBV latent proteins and to the more common CTL responses that cross-react against type A and type B virus isolates. IMAGES:

EB病毒（Epstein-Barr virus，EBV）是一类具备细胞生长转化潜能的嗜B淋巴细胞因子，其感染引发的潜在致病效应可在健康病毒携带者体内通过病毒特异性细胞毒性T淋巴细胞（CTL）监视得到有效控制。目前此类CTL应答所靶向的抗原尚未明确，但这类抗原大概率源自病毒转化B细胞中组成性表达的一种或多种EB病毒“潜伏”蛋白。本研究分析了两名EB病毒免疫供体的CTL应答靶标特异性，这两名供体仅对A型而非B型病毒分离株转化的自体细胞表现出优先反应性。我们将四种在A、B型病毒间存在等位基因多态性的EB病毒潜伏蛋白的编码序列——即EB病毒核抗原（EBV nuclear antigens，EBNAs）EBNA 2、EBNA 3a、EBNA 3c及EBNA先导蛋白——置于痘苗病毒启动子P7.5的调控下插入痘苗病毒载体，用于在合适的靶细胞中表达相关EBNA蛋白。据此，我们将第一名供体的CTL应答定位至A型EBNA 2蛋白，第二名供体的CTL应答则定位至A型EBNA 3a蛋白。后续我们构建了一系列在A型EBNA 2编码序列内携带特定内部缺失的重组痘苗病毒；通过使用这些载体，上述A型EBNA 2特异性CTL应答被证实靶向该蛋白N端附近一段含100个氨基酸残基的片段内的表位。本研究明确证实了EB病毒系统中人CTL对病毒编码核抗原的识别；此外，本研究建立了一套可推广至所有EB病毒潜伏蛋白，以及适用于更常见的、能与A、B型病毒分离株发生交叉反应的CTL应答的实验方法。图像：