Muthumani Karuppiah, Zhang Donghui, Dayes Nathanael S, Hwang Daniel S, Calarota Sandra A, Choo Andrew Y, Boyer Jean D, Weiner David B
Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Virology. 2003 Sep 15;314(1):134-46. doi: 10.1016/s0042-6822(03)00459-8.
HIV-1 sequences are highly diverse due to the inaccuracy of the viral reverse transcriptase. This diversity has been studied and used to categorize HIV isolates into subtypes or clades, which are geographically distinct. To develop effective vaccines against HIV-1, immunogens representing different subtypes may be important for induction of cross-protective immunity, but little data exist describing and comparing the immunogenicity induced by different subtype-based vaccines. This issue is further complicated by poor expression of HIV structural antigens due to rev dependence. One costly approach is to codon optimize each subtype construct to be examined. Interestingly, cis-acting transcriptional elements (CTE) can also by pass rev restriction by a rev independent export pathway. We reasoned that rev+CTE constructs might have advantages for such expression studies. A subtype A envelope sequence from a viral isolate from east Africa was cloned into a eukaryotic expression vector under the control of the CMV-IE promoter. The utility of inclusion of the Mason-Pfizer monkey virus (MPV)-CTE with/without rev for driving envelope expression and immunogenicity was examined. Expression of envelope (gp120) was confirmed by immunoblot analysis and by pseudotype virus infectivity assays. The presence of rev and the CTE together increased envelope expression and viral infection. Furthermore the CTE+rev construct was significantly more immunogenic then CTE alone vector. Isotype analysis and cytokine profiles showed strong Th1 response in plasmid-immunized mice, which also demonstrated the superior nature of the rev+CTE construct. These responses were of similar or greater magnitude to a codon-optimized construct. The resulting cellular immune responses were highly cross-reactive with a HIV-1 envelope subtype B antigen. This study suggests a simple strategy for improving the expression and immunogenicity of HIV subtype-specific envelope antigens as plasmid or vector-borne immunogens.
由于病毒逆转录酶的不准确性，HIV-1序列具有高度多样性。这种多样性已被研究并用于将HIV分离株分类为亚型或进化枝，它们在地理上是不同的。为了开发针对HIV-1的有效疫苗，代表不同亚型的免疫原对于诱导交叉保护性免疫可能很重要，但描述和比较不同基于亚型的疫苗所诱导的免疫原性的数据很少。由于对rev的依赖性，HIV结构抗原的表达不佳使这个问题更加复杂。一种成本高昂的方法是对每个要检测的亚型构建体进行密码子优化。有趣的是，顺式作用转录元件（CTE）也可以通过独立于rev的输出途径绕过rev限制。我们推断，rev+CTE构建体在这种表达研究中可能具有优势。将来自东非病毒分离株的A亚型包膜序列克隆到受CMV-IE启动子控制的真核表达载体中。研究了包含/不包含rev的猴空泡病毒（MPV）-CTE对驱动包膜表达和免疫原性的效用。通过免疫印迹分析和假型病毒感染性测定证实了包膜（gp120）的表达。rev和CTE共同存在增加了包膜表达和病毒感染。此外，CTE+rev构建体的免疫原性明显强于单独的CTE载体。同型分析和细胞因子谱显示，在质粒免疫的小鼠中存在强烈的Th1反应，这也证明了rev+CTE构建体的优越性。这些反应与密码子优化构建体的反应相似或更强。产生的细胞免疫反应与HIV-1包膜B亚型抗原具有高度交叉反应性。这项研究提出了一种简单的策略，可提高HIV亚型特异性包膜抗原作为质粒或载体携带的免疫原的表达和免疫原性。
