Rodriguez F, An L L, Harkins S, Zhang J, Yokoyama M, Widera G, Fuller J T, Kincaid C, Campbell I L, Whitton J L
Department of Neuropharmacology, The Scripps Research Institute, La Jolla, California 92037, USA.
J Virol. 1998 Jun;72(6):5174-81. doi: 10.1128/JVI.72.6.5174-5181.1998.
Our previous studies have shown that isolated cytotoxic T lymphocyte (CTL), B-cell, and T-helper epitopes, for which we coined the term minigenes, can be effective vaccines; when expressed from recombinant vaccinia viruses, these short immunogenic sequences confer protection against a variety of viruses and bacteria. In addition, we have previously demonstrated the utility of DNA immunization using plasmids encoding full-length viral proteins. Here we combine the two approaches and evaluate the effectiveness of minigenes in DNA immunization. We find that DNA immunization with isolated minigenes primes virus-specific memory CTL responses which, 4 days following virus challenge, appear similar in magnitude to those induced by vaccines known to be protective. Surprisingly, this vigorous CTL response fails to confer protection against a normally lethal virus challenge, although the CTL appear fully functional because, along with their high lytic activity, they are similar in affinity and cytokine secretion to CTL induced by virus infection. However this DNA immunization with isolated minigenes results in a low CTL precursor frequency; only 1 in approximately 40,000 T cells is epitope specific. In contrast, a plasmid encoding the same minigene sequences covalently attached to the cellular protein ubiquitin induces protective immunity and a sixfold-higher frequency of CTL precursors. Thus, we show that the most commonly employed criterion to evaluate CTL responses-the presence of lytic activity following secondary stimulation-does not invariably correlate with protection; instead, the better correlate of protection is the CTL precursor frequency. Recent observations indicate that certain effector functions are active in memory CTL and do not require prolonged stimulation. We suggest that these early effector functions of CTL, immediately following infection, are critical in controlling virus dissemination and in determining the outcome of the infection. Finally, we show that improved performance of the ubiquitinated minigenes most probably requires polyubiquitination of the fusion protein, suggesting that the enhancement results from more effective delivery of the minigene to the proteasome.
我们之前的研究表明，我们创造了“微型基因”这一术语的分离细胞毒性T淋巴细胞（CTL）、B细胞和辅助性T细胞表位可以成为有效的疫苗；当从重组痘苗病毒中表达时，这些短免疫原性序列可提供针对多种病毒和细菌的保护。此外，我们之前已经证明了使用编码全长病毒蛋白的质粒进行DNA免疫的效用。在此，我们将这两种方法结合起来，并评估微型基因在DNA免疫中的有效性。我们发现，用分离的微型基因进行DNA免疫可引发病毒特异性记忆CTL反应，在病毒攻击4天后，其强度与已知具有保护作用的疫苗诱导的反应相似。令人惊讶的是，尽管CTL看起来功能完全正常，因为它们除了具有高裂解活性外，在亲和力和细胞因子分泌方面与病毒感染诱导的CTL相似，但这种强烈的CTL反应未能提供针对正常致死性病毒攻击的保护。然而，用分离的微型基因进行这种DNA免疫导致CTL前体频率较低；大约每40,000个T细胞中只有1个是表位特异性的。相比之下，编码与细胞蛋白泛素共价连接的相同微型基因序列的质粒可诱导保护性免疫和CTL前体频率提高六倍。因此，我们表明，评估CTL反应最常用的标准——二次刺激后裂解活性的存在——并不总是与保护相关；相反，与保护更好相关的是CTL前体频率。最近的观察表明，某些效应功能在记忆CTL中是活跃的，并且不需要长时间刺激。我们认为，CTL在感染后立即发挥的这些早期效应功能对于控制病毒传播和确定感染结果至关重要。最后，我们表明，泛素化微型基因性能的改善很可能需要融合蛋白的多聚泛素化，这表明这种增强是由于微型基因更有效地递送至蛋白酶体所致。
