Protective CD8+ T cell responses and role of viral escape in HBV infection

病毒特异性CD8+T细胞在乙肝病毒控制中起着核心作用。然而，在慢性乙肝感染中，病毒特异性CD8+T细胞被证明是功能受损的。尽管这些被广泛接受的发现，在乙肝免疫生物学的核心问题还没有得到解决。具体地说，CD8+T细胞表位在乙肝病毒感染的结局中可能具有保护性作用还没有确定，病毒逃逸在乙肝病毒持续存在中的作用仍然存在争议。事实上，只有少数几个乙肝病毒特异的CD8+T细胞表位已被确定，主要受人类白细胞抗原A2的限制。分析这些HLA-A2表位的研究表明，病毒逃逸在乙肝病毒特异性T细胞衰竭中不起主要作用。然而，针对人类病毒(如HIV和丙型肝炎病毒)的保护性CD8+T细胞反应通常受到对病毒施加最强选择压力的A2(例如B27、B57、A3)以外的HLA等位基因的限制。我们以前已经确定了第一个人类白细胞抗原-B27限制性的乙肝病毒特异性CD8+T细胞表位，并获得了人类白细胞抗原-B27驱动的病毒逃逸的证据。这些结果表明，人类白细胞抗原B27可能在乙肝病毒感染中起主导和保护作用，而病毒逃逸确实是导致病毒特异性T细胞衰竭的核心机制，从而质疑目前该领域中病毒逃逸并不在乙肝病毒感染中起主要作用的概念。为了扩展这些初步发现并在更广泛的范围内证实它们，我们的目标是识别更多受可能保护性等位基因如HLA-B27、B57和A3限制的主导的乙肝病毒特异性CD8+T细胞表位。这些研究将在一大批急性、缓解或慢性乙肝病毒感染的患者中进行。这一结果将克服目前对乙肝免疫发病机制的有限理解，即局限于人类白细胞抗原A2背景。通过分析与新发现的主要CD8+T细胞表位相对应的病毒序列，我们将阐明病毒逃逸在持续的乙肝病毒感染中的作用。在一个大的患者队列中进行全基因组乙肝病毒序列分析将允许在已识别的表位之外识别与人类白细胞抗原相关的序列多态。由于功能限制，如重叠的读框，被认为强烈限制病毒逃逸，我们将在细胞培养模型中分析已识别的病毒逃逸突变(例如，那些被识别为HLA-B27的突变)对乙肝病毒复制的影响。建立表达人类白细胞抗原B27的细胞培养系统，可以分析病毒逃逸对病毒特异性CD8+T细胞控制乙肝病毒的影响。最后，由于病毒逃逸被描述为在HIV和HBV感染中对CD8+T细胞的表型和功能有重要影响，我们将分析针对野生型或突变表位的HBV特异性CD8+T细胞的表型和功能。综上所述，我们的结果将有助于更好地定义保护性的HBV特异性CD8+T细胞，阐明病毒逃逸在HBV持久化中的作用，并可能改变目前公认的HBV免疫生物学概念。

Virus-specific CD8+ T cells play a central role in HBV control. In chronic HBV infection, however, virusspecific CD8+ T cells have been shown to be functionally impaired. Despite these well accepted findings, central questions in HBV immunobiology have not been addressed. Specifically, CD8+ T cell epitopes that might be protective in outcome of HBV infection have not been defined and the role of viral escape in HBV persistence remains controversial. Indeed, only few HBV-specific CD8+ T cell epitopes, mostly restricted by HLA-A2, have been identified. Studies analyzing these HLA-A2 epitopes have suggested that viral escape does not play a major role in HBV-specific T cell failure. However, protective CD8+ T cell responses against human viruses such as HIV and HCV are typically restricted by HLA alleles other than A2 (e.g. B27, B57, A3) that exert the strongest selective pressure on the virus. We have previously identified the first HLA-B27 restricted HBV-specific CD8+ T cell epitopes and obtained evidence for HLA-B27 driven viral escape. These results indicate that HLA-B27 may have a dominant and protective role in HBV infection and that viral escape is indeed a central mechanism of HBV-specific T cell failure, thus questioning the current concept in the field that viral escape does not play a major role in HBV infection.To extend these preliminary findings and to confirm them on a broader scale, we aim to identify additional dominant HBV-specific CD8+ T cell epitopes restricted by likely protective HLA alleles such as HLA-B27, B57, and A3. These studies will be performed in a large cohort of patients with acute, resolved, or chronic HBV infection. The results will overcome the currently limited understanding of HBV immunopathogenesis that is restricted to the HLA-A2 background. By analyzing viral sequences corresponding to newly identified dominant CD8+ T cell epitopes, we will clarify the role of viral escape in persistent HBV infection. A fullgenome HBV sequence analysis in a large patient cohort will allow the identification of HLA-associated sequence polymorphisms outside of identified epitopes. Since functional constraints, e.g. overlapping reading frames, are thought to strongly limit viral escape, we will analyze the impact of identified viral escape mutations (e.g. those identified for HLA-B27) on HBV replication in a cell culture model. Establishment of an HLA-B27 expressing cell culture system will allow the analysis of the effect of viral escape on HBV control by virus-specific CD8+ T cells. Finally, since viral escape has been described to have an important impact on CD8+ T cell phenotype and function in HIV and HBV infection, we will analyze the phenotype and function of HBV-specific CD8+ T cells targeting wild-type or mutated epitopes.In sum, our results will contribute to a better definition of protective HBV-specific CD8+ T cells, clarify the role of viral escape in HBV persistence, and may change currently accepted concepts of HBV immunobiology.