FUNCTIONAL SIGNIFICANCE OF CTL ESCAPE

CTL 转义的功能意义

• 批准号: 8173086
• 负责人: David I Watkins
• 金额: $ 5.16万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173086
• 关键词: Acute; Affect; Alleles; Animal Model; Animals; Antigens; Arginine; Ascaridil; Binding; CD8-Positive T-Lymphocytes; CD8B1 gene; Cohort Studies; Computer Retrieval of Information on Scientific Projects Database; Development; Epitopes; Evolution; Exhibits; Frequencies; Funding; Genetic Services; Goals; Grant; HIV; HIV vaccine; HLA-B 2705 antigen; Histocompatibility Antigens Class I; Human; Immunodominant Epitopes; Immunologic Deficiency Syndromes; Immunology; In Vitro; Individual; Infection; Institution; Intention; Knowledge; Lead; MHC Class I Genes; Macaca; Macaca mulatta; Maintenance; Maps; Mediator of activation protein; Memory; Modeling; Mutate; Mutation; Outcome; Peptides; Phase; Plasma; Positioning Attribute; Process; Relative (related person); Research; Research Personnel; Resources; SIV; Services; Source; System; T cell response; T-Lymphocyte; T-Lymphocyte Epitopes; Testing; United States National Institutes of Health; Vaccinated; Vaccine Design; Variant; Viral; Viral Load result; Virus; Virus Replication; bean; fitness; in vivo; mutant; research study; response; virology

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. Objective: To evaluate the impact of AIDS virus evolution on the design of vaccines, we will determine whether mutations that facilitate AIDS virus escape from CD8+ T-cell responses also affect viral fitness in vitro and in vivo. We are also attempting to determine the major factors that contribute to elite control of AIDS virus replication. We have recently developed an animal model of elite control in Mamu-B*08+ Indian rhesus macaques. Remarkably, 50% of Mamu-B*08+ Indian rhesus macaques control replication of SIV, and Mamu-B*08 and HLA-B*27 bind similar peptides. Thus, these Mamu-B*08+ macaques are ideal for modeling human ECs. Understanding why ECs suppress viral replication should facilitate the development of an effective HIV vaccine. Elite controllers of HIV and SIV are studied with the goal of understanding immunological mechanisms underlying successful control of immunodeficiency virus replication. Elite control is associated with particular MHC class I alleles, implicating CD8+ T cells as mediators of control. Since in vivo CD8+ cell depletion in two Mamu-B*08+ EC rhesus macaques results in an immediate increase in viral replication, CD8+ lymphocytes are likely important for control in these ECs. Interestingly, SIVmac239¿nef-vaccinated Mamu-B*08+ macaques exhibit almost complete control of a heterologous SIVsmE660 challenge. Additionally, HLA-B*27 and Mamu-B*08 bind the same peptides requiring an arginine at position 2, and antigen-specific Mamu-B*08-restricted CD8+ T cell responses dominate the acute phase in ECs. These results implicate MHC class I-bound peptide-specific memory CD8+ T lymphocytes in control of viral replication in Mamu-B*08+ rhesus macaques. Our initial experiment using an 8X mutant virus containing viral escape mutations in all of the B*08-restricted immunodominant epitopes, demonstrated that CD8 T cell responses are important in B*08-associated elite control. At week 14 post-infection the geometric mean viral load for ten Mamu-B*08+ animals infected with SIVmac239 was 5,500 vRNA copy Eq/ml plasma, whereas the group of ten animals infected with 8X-SIVmac239 had a geometric mean viral load of 101,000 vRNA copy Eq/ml plasma. In spite of this significant difference, two of the ten 8X animals became ECs. In addition, the 8X-infected animals made substantially more frequent responses to the subdominant B*08-restricted epitopes that were not mutated in the 8X construct. Some 8X animals also responded, albeit much less frequently than the wild-type infected animals, to four of the eight mutated epitopes. We have therefore created a new mutant virus construct, termed 12X, that both introduces escape mutations into the previously non-mutated subdominant epitopes and re-introduces "better" escape mutations into the epitopes that exhibited breakthrough in the 8X experiment. We are presently in the process of making and testing this virus in vitro. It is then our intention to introduce the 12X virus into 10 non-B*08 animals to completely evaluate the in vivo fitness of the new construct before infecting another group of B*08+ macaques. We have also initiated a new direction as a part of this project systematically investigating the outcomes of various CD8 T cell escape mutations in viruses transmitted to individuals that do not harbor the restricting MHC allele for the particular mutation. Recent evidence from human cohort studies suggests that the implication of CTL escape may be the evolution of HIV to avoid specific responses, and perhaps even the loss of particular MHC/elite control associations. Our group and others have demonstrated that escape mutation containing viruses that are transmitted to individuals without the particular selecting allele can either revert to wild-type sequence or maintain themselves in the new host. The relative frequency of both scenarios must be known to determine if HIV will or will not evolve to evade particular T cell responses, as only the later instance will lead to HIV/SIV evolution. This is nearly impossible in the HIV system, as the sequence of the infecting virus is rarely known. We have systematically mapped both T cell epitopes and T cell escape mutations in SIVmac239 for several common MHC class I alleles in our rhesus macaque system. We are in the process of constructing systematic escape epitope viruses for the A*01, A*02, A*11, B*08 and B*17 class I alleles. We will introduce these viruses into animals that lack the selecting class I alleles and determine the frequency of escape reversion and maintenance. With this knowledge, we will make quantitative conclusions about the implications of CTL escape for various MHC class I alleles, including those associated with elite control. We are presently constructing and testing these viruses in vitro, but intend to complete these studies during the next year. This research used WNPRC Animal Services, Genetics Services, and Immunology & Virology Services. PUBLICATIONS: Valentine LE, Loffredo JT, Bean AT, Le¿n EJ, MacNair CE, Beal DR, Piaskowski SM, Klimentidis YC, Lank SM, Wiseman RW, Weinfurter JT, May GE, Rakasz EG, Wilson NA, Friedrich TC, O'Connor DH, Allison DB, Watkins DI. Infection with "escaped" virus variants impairs control of simian immunodeficiency virus SIVmac239 replication in Mamu-B*08-positive macaques. J Virol. 2009 Nov; 83(22):11514-27. Epub 2009 Sep 2. PMID: 19726517 Loffredo JT, Sidney J, Bean AT, Beal DR, Bardet W, Wahl A, Hawkins OE, Piaskowski S, Wilson NA, Hildebrand WH, Watkins DI, Sette A. Two MHC class I molecules associated with elite control of immunodeficiency virus replication, Mamu-B*08 and HLA-B*2705, bind peptides with sequence similarity. J Immunol. 2009 Jun 15; 182(12):7763-75. PMID: 19494300

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以出现在其他 CRISP 条目中 列出的机构是。 对于中心来说，它不一定是研究者的机构。 目的：为了评估艾滋病病毒进化对疫苗设计的影响，我们将确定促进艾滋病病毒逃避 CD8+ T 细胞反应的突变是否也会影响病毒的体外和体内适应性。我们还试图确定主要的影响因素。我们最近在 Mamu-B*08+ 印度恒河猴中开发了一种精英控制动物模型，值得注意的是，50% 的 Mamu-B*08+ 印度恒河猴。猕猴控制 SIV 的复制，并且 Mamu-B*08 和 HLA-B*27 结合相似的肽，因此，这些 Mamu-B*08+ 猕猴是模拟人类 EC 的理想选择，了解 EC 抑制病毒复制的原因应有助于发展 SIV。有效的艾滋病毒疫苗。 研究 HIV 和 SIV 的精英控制者的目的是了解成功控制免疫缺陷病毒复制的免疫机制，精英控制与特定的 MHC I 类等位基因相关，这表明 CD8+ T 细胞作为控制的介质。两只 Mamu-B*08+ EC 恒河猴导致病毒复制立即增加，CD8+ 淋巴细胞可能对于这些 EC 的控制很重要。 SIVmac239¿ nef 疫苗接种的 Mamu-B*08+ 猕猴表现出对异源 SIVsmE660 攻击的几乎完全控制。此外，HLA-B*27 和 Mamu-B*08 结合需要 2 位精氨酸和抗原特异性 Mamu- 的相同肽。 B*08 限制性 CD8+ T 细胞反应在 EC 的急性期中占主导地位。这些结果表明 MHC I 类结合肽特异性记忆。 CD8+ T 淋巴细胞控制 Mamu-B*08+ 恒河猴中的病毒复制。 我们最初的实验使用在所有 B*08 限制性免疫显性表位中含有病毒逃逸突变的 8X 突变病毒，证明 CD8 T 细胞反应在 B*08-精英相关对照中很重要。在感染后第 14 周，几何平均值呈几何平均值。感染 SIVmac239 的 10 只 Mamu-B*08+ 动物的病毒载量为 5,500 vRNA 拷贝 Eq/ml 血浆，而感染 SIVmac239 的 10 只动物组的病毒载量为8X-SIVmac239 的几何平均病毒载量为 101,000 vRNA 拷贝 Eq/ml 血浆，尽管存在这种显着差异，但 10 只 8X 动物中的两只变成了 EC。此外，8X 感染的动物对次优势动物做出了明显更频繁的反应。 8X 构建体中未突变的 B*08 限制性表位 一些 8X 动物也有反应，但频率比野生型感染动物低得多。因此，我们创建了一种新的突变病毒构建体，称为 12X，它既将逃逸突变引入到先前未突变的次优势表位中，又将“更好的”逃逸突变重新引入到表现出突破性的表位中。 8X 实验目前正在体外制备和测试该病毒，然后我们打算将 12X 病毒引入 10 只非 B*08 动物中以全面评估该病毒。在感染另一组 B*08+ 猕猴之前，测试新构建体的体内适应性。 作为该项目的一部分，我们还启动了一个新方向，系统地研究传播给不携带特定突变的限制性 MHC 等位基因的个体的各种 CD8 T 细胞逃逸突变的结果。来自人类队列研究的最新证据表明， CTL 逃逸的含义可能是 HIV 的进化，以避免特定的反应，甚至可能是特定 MHC/精英控制关联的丧失。我们的小组和其他人已经证明，包含病毒的逃逸突变会传播给没有特定选择等位基因的个体。可以恢复为野生型必须知道这两种情况的相对频率，以确定 HIV 是否会进化以逃避特定的 T 细胞反应，因为只有后者才会导致 HIV/SIV 进化。在 HIV 系统中这是不可能的，因为感染病毒的序列很少为人所知，我们已经系统地绘制了恒河猴系统中几个常见 MHC I 类等位基因的 SIVmac239 中的 T 细胞表位和 T 细胞逃逸突变。构建针对 A*01、A*02、A*11、B*08 和 B*17 I 类等位基因的系统逃逸表位病毒的过程 我们将把这些病毒引入缺乏 I 类选择等位基因的动物中，并确定其有了这些知识，我们将对 CTL 逃逸对各种 MHC I 类等位基因（包括与精英控制相关的等位基因）的影响做出定量结论。明年完成这些研究。 这项研究使用了 WNPRC 动物服务、遗传学服务以及免疫学和病毒学服务。 出版物： 瓦伦丁 LE、洛弗雷多 JT、比恩 AT、Le¿ n EJ、MacNair CE、Beal DR、Piaskowski SM、Klimentidis YC、Lank SM、Wiseman RW、Weinfurter JT、May GE、Rakasz EG、Wilson NA、Friedrich TC、O'Connor DH、Allison DB、Watkins DI。 “逃逸”病毒变种损害了猿猴免疫缺陷病毒 SIVmac239 复制的控制Mamu-B*08-阳性猕猴。2009 年 11 月；83(22):11514-27。Epub 2009 年 9 月 2 日。PMID：19726517 Loffredo JT、Sidney J、Bean AT、Beal DR、Bardet W、Wahl A、Hawkins OE、Piaskowski S、Wilson NA、Hildebrand WH、Watkins DI、Sette A。两种与免疫缺陷病毒复制的精英控制相关的 MHC I 类分子， Mamu-B*08 和 HLA-B*2705 结合具有序列相似性的肽，2009 年 6 月。 15；182(12)：7763-75。