IDENTIFICATION OF ORTHOPOXVIRUS-DERIVED CD8+ T CELL EPITOPES MACAQUES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。牛痘病毒疫苗接种诱导强烈的病毒特异性CD8+ T细胞反应，在控制痘病毒感染中起重要作用。然而，对于作为牛痘特异性CD8+ T细胞靶标的特定病毒蛋白知之甚少，并且在编码超过200个开放阅读框（orf）的基因组中鉴定T细胞表位是一项特别具有挑战性的任务。作为鉴定免疫显性痘病毒蛋白的一种方法，我们使用了一种算法来预测能够结合猕猴常见MHC I类分子Mamu-A*01的牛痘肽。我们从97个牛痘orf中合成了294个多肽，对这些多肽进行筛选，以供接种疫苗的猕猴T细胞识别，并鉴定出免疫原性牛痘蛋白。这些肽的选择是基于它们的高预测结合效率和痘苗蛋白质组的总体代表。使用IFNgamma elisa法对两周前接种Dryvax的Mamu-A*01+猕猴的pbmc进行细胞免疫应答的初步评估。这些猕猴对多种正痘病毒抗原产生了相对较强的反应：每只猕猴识别6-12个池，其中4个池被两只猕猴识别。接下来，我们对这些阳性池进行解卷积，并测试接种疫苗的动物对单个肽的反应。接种疫苗的猕猴能够识别来自8种不同orf的10个单肽。与其他正痘病毒序列的比较表明，这些表位高度保守，存在于牛痘、天花和猴痘中。这些结果表明，病毒特异性CD8+ T细胞反应广泛针对多种牛痘蛋白，并且这些T细胞表位的一个子集在正痘病毒中高度保守。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Vaccination with vaccinia virus induces a vigorous virus-specific CD8+ T cell response that plays an important role in control of poxvirus infection. However, little is known about the specific viral proteins that are serve as targets of vaccinia-specific CD8+ T cells, and identification of T cell epitopes in a genome that encodes over 200 open reading frames (ORFs) is a particularly challenging task. As one approach to the identification of immunodominant poxvirus proteins, we used an algorithm for the prediction of vaccinia peptides able to bind to the common macaque MHC class I molecule Mamu-A*01. We synthesized 294 peptides derived from 97 vaccinia ORFs, screened these peptides for recognition by T cells from vaccinated macaques and identified immunogenic vaccinia proteins. These peptides were chosen based on their high predicted binding efficiency and overall representation of the vaccinia proteome. The initial assessment of cellular immune responses were performed using IFNgamma ELISPO assays on PBMCs from Mamu-A*01+ macaques that were vaccinated two weeks previously with Dryvax. These macaques developed relatively strong responses against multiple orthopoxvirus antigens: 6-12 pools were recognized per animal, with 4 pools being recognized by both macaques. We next deconvoluted these positive pools and tested our vaccinated animals for responses to individual peptides. Vaccinated macaques recognized 10 single peptides from 8 different ORFs. Comparison with other orthopoxvirus sequences revealed that these epitopes were highly conserved and present in vaccinia, variola, and monkeypox. These results suggest that the virus-specific CD8+ T cell response is broadly directed against multiple vaccinia proteins and that a subset of these T cell epitopes are highly conserved among orthopoxviruses.