Antigen stability as a parameter in rational T(CD8+)-targeting vaccine design

描述(申请人提供)：CD8+T细胞(TCD8+)通过清除表面显示病毒的感染细胞，在限制病毒传播方面发挥关键作用 衍生的多肽与主要组织相容性复合体I类(MHCI)分子结合。由于许多病毒在感染后数小时内复制，有效的TCD8+作用依赖于病毒蛋白质合成后不久的分解代谢，其机制尚不清楚。我们对这些机制的探索揭示了多肽的起始显示在细胞表面，因此，多肽/MHCI复合体的密度根据抗原的性质而有很大的不同。因此，没有有效转位到内质网的胞浆蛋白和信号序列(SS)蛋白出现得很快，而高效转位蛋白的处理却大大延迟。此外，无屏蔽的疏水性(如无连接的跨膜区)构成了足够快速的蛋白质分解代谢的一个驱动力。基于这些观察，我们假设TCD8+针对来自最快速降解的蛋白质的表位具有最强的保护作用，这是因为病毒在感染后的早期时间点中断了复制。矛盾的是，我们预测这些相同的蛋白质在启动TCD8+反应方面表现较差，因为交叉呈递过程--即抗原从感染细胞转移到树突状细胞--取决于蛋白质的稳定性。通过延伸，我们提出了一种以TCD8+为靶向的疫苗的开发策略，该策略包括：首先，鉴定一种病毒蛋白，该病毒蛋白在其自然状态下与快速多肽生成相关，从而更有效地清除病毒。在第二步中，抗原被设计成在疫苗表达时是稳定的，以优化交叉呈现。这些想法将通过体外和体内实验相结合的方式进行验证，使用一组主要由重组小鼠病原体--重组皮肤病病毒和重组腺病毒表达的模型抗原，如果需要异源表达的话。这些实验的结果可能从根本上改变TCD8+靶向疫苗的合理设计，这是一个与许多人类病原体相关的目标，包括艾滋病毒、疱疹病毒和流感。

DESCRIPTION (provided by applicant): CD8+ T cells (TCD8+) play critical roles in limiting viral spread through the elimination of virus-infected cells, which display at their surfaces virus derived peptides in combination with major histocompatibility complex class I (MHCI) molecules. Since many viruses replicate within hours after infection, effective TCD8+ action depends upon catabolism of viral proteins shortly after their synthesis through mechanisms that remain poorly understood. Our pursuit of these mechanisms has revealed that onset of peptide display at the cell surface and, consequently, the density of peptide/MHCI complexes varies considerably depending upon properties of the antigen. Thus, cytosolic proteins and signal sequence (SS)-bearing proteins that are not efficiently translocated into the endoplasmic reticulum appear rapidly while the processing of efficiently translocated proteins is substantially delayed. Furthermore, unshielded hydrophobicity (such as an untethered transmembrane domain) constitutes one driver of sufficiently rapid protein catabolism. Based upon these observations, we hypothesize that TCD8+ specific for epitopes derived from the most rapidly degraded proteins are most protective due to interruption of viral replication at early time points followin infection. Paradoxically, we predict that these same proteins are inferior in priming for a TCD8+ response, since the process of cross-presentation - in which antigen is transferred from an infected cell to the dendritic cell - depends upon stability of the protein. By extension, we propose a strategy for development of a TCD8+-targeting vaccine that involves, first, the identification of a viral protein which, in its natural state, is associated with rapid peptide generation and, therefore more efficient viral clearance. In second step, the antigen is engineered to be stable when expressed by a vaccine, in order to optimize cross- presentation. These ideas will be tested with a combination of in vitro and in vivo experiments using a panel of model antigens expressed primarily by recombinant ectromelia virus, a natural mouse pathogen, and recombinant adenovirus when heterologous expression will be necessary. Results of these experiments could fundamentally alter the rational design of TCD8+-targeting vaccines, a goal with relevance to many human pathogens, including HIV, herpesviruses, and influenza.