Non-Human Primate Discovery Team

非人类灵长类动物发现团队

• 批准号: 8294658
• 负责人: NORMAN L LETVIN
• 金额: $ 219.1万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8294658
• 关键词: AIDS/HIV problem; Acute; Address; Anatomy; Animals; Antibodies; Attenuated Live Virus Vaccine; B-Lymphocyte Subsets; B-Lymphocytes; Biological; Biological Assay; Blood; Blood Cells; Blood Circulation; CCR5 gene; CD3 Antigens; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; Cell Count; Cells; Chronic; Clinical; Cohort Studies; Complement; Containment; Data; Development; Evaluation; Event; Evolution; FCGR3B gene; Frequencies; Gagging; Generations; Genetic; HIV-1; Human; Immune; Immune response; Immunologics; Immunology; In Vitro; Individual; Infection; Infusion procedures; Kinetics; Lymphocyte Count; Lymphocyte Function; Lymphoid Tissue; MS4A1 gene; Macaca mulatta; Mediating; Memory; Methods; Modeling; Monitor; Monkeys; Monoclonal Antibodies; Monoclonal Antibody CD20; Mus; Natural Immunity; Natural Killer Cells; Nature; Pathogenesis; Pilot Projects; Plasma; Plasma Cells; Play; Primate Lentiviruses; Process; Property; RNA; Regimen; Reporting; Role; Route; SIV; Sampling; Seminal; Shapes; Staining method; Stains; Subfamily lentivirinae; System; T-Lymphocyte; Time; Tissues; Tropism; V3 Loop; Vaccination; Vaccines; Variant; Vertebral column; Viral; Viral Load result; Virus; cohort; cytokine; env Genes; enzyme linked immunospot assay; fitness; gastrointestinal; immune function; in vivo; lymph nodes; memory CD4 T lymphocyte; nonhuman primate; novel vaccines; peripheral blood; pressure; receptor; rectal; response; simian human immunodeficiency virus; superinfection; tool; transmission process; vaccine candidate; viral RNA

The contribution of innate immunity to the early containment of HIV-1 replication remains unknown. We have initiated studies to evaluate the role of NK cells in primate lentivirus control in a 10 monkey study in which NK cells have been eliminated from the circulation of animals by infusion of an anti-CD16 monoclonal antibody. In this ongoing study we have demonstrated that elimination of NK cells (CD3-, CD20-, NKG2+ cells) from the peripheral blood of monkeys for 7 days does not effect the kinetics of plasma viral RNA levels or loss of memory CD4+ T lymphocytes in the first weeks following an infection with SIVmac251. This observation suggests that NK cells are playing a negligible role in the early control of SIVmac251 replication. The observation that HIV-1 envelope sequences evolve over time in infected humans to escape from antibody neutralization suggests that humoral immune responses may be contributing to viral control and, in the process, exerting selection pressure on the virus. We have recently optimized the administration regimen of an anti- CD20 monoclonal antibody to eliminate B lymphocytes for up to a year from the peripheral blood and lymph nodes of rhesus monkeys. We are using this antibody as a tool in an ongoing 20 monkey study to evaluate the contribution of antibody to early containment of SIVmac251 infection. One hundred days following infection, the anti-CD20 infused monkeys still do not have detectable peripheral blood B lymphocytes. Plasma viral RNA levels from these animals are still pending. However, we have not observed a significant difference in peripheral blood CD4+ T cell numbers and SIV-gag ELISPOT responses between the monkeys that received the experimental and the control monoclonal antibody infusions, suggesting that viral replication during this period of time is not significantly different in the experimental and control monkeys. We will initiate a complementing study in May, 2007 to deplete B cells in the setting of SIVmac239 delta nef vaccination and SIVmac251 challenge to assess the contribution of antibody generation to the protection conferred by this live attenuated vaccine. Although the frequency of HIV-1 superinfection is not known, there is increasing evidence that individuals already infected with HIV-1 can be infected with a heterologous strain of the virus. This clinical setting may provide an important window through which to view the immune and virologic correlates of protection against HIV-1 infection. The SIV-infected rhesus monkey model provides a powerful system for studying events associated with lentivirus superinfection. This model provides a means of controlling potential variables that might impact on transmission, such as the genetic relatedness of the two viruses, the route and timing of virus exposure, and the immune status of the previously infected individual. We have created a model for superinfection in monkeys using two SIV isolates that are as disparate in sequence as any two circulating HIV-1 isolates. We will also include in these studies cohorts of monkeys that have received received nef-deleted SIVmac239 virus so that the degree of protection afforded by a prior infection with replication-competent virus can be compared with that provided by a live attenuated virus vaccine. We have initiated studies in this nonhuman primate model of lentivirus superinfection. To differentiate and quantify SIVmac251 and SIVsmE660 in the blood, we have developed a quantitative PCR method for determining the RNA levels of the two virus strains in plasma samples. We have established two cohorts of rhesus monkeys, one infected with SIVmac251 and the other with SIVsmE660. We have defined the baseline immune function of these monkeys by characterizing their CD4+ and CD8+ T lymphocyte memory subsets and their SIV-specific T lymphocyte function by Elispot assay and intracellular cytokine staining. These monkeys were then exposed to the heterologous virus via 6 weekly intrarectal challenges. Their CD4+ T lymphocyte memory subsets, total CD4+ T lymphocyte counts, and plasma viral loads were assessed prospectively. At the completion of 6 intrarectal challenges, these animals had no evidence of superinfection as determined by quantitative PCR. We will monitor these animals for an additional four weeks, and if there is no evidence of the second virus in their blood, we will expose them to another round of 6 intrarectal challenges. If these monkeys remain protected against superinfection, we will begin studies to determine the mechanism mediating this protection. The first of these studies will be to assess the role of CD8+ T lymphocytes in this protection by depleting these cells through monoclonal antibody infusion and assessing the protection afforded by the prior infection. It will be essential to define the nature of the transmitted virus to create an effective HIV-1 vaccine. A report by Hunter and colleagues suggested that the transmitted virus may have shortened variable loops. To elucidate whether deletions in the variable loops of Env reflect a transmission bottleneck, we are examining viral sequence evolution in a cohort of 5 rhesus monkeys that were infected with a genetically-defined stock of SIVmac251 via repeated intrarectal inoculation. At the time of peak viral replication and up to 6 months after infection, we did not observe variable loop deletions in the env gene. Interestingly, as these monkeys progressed from acute to chronic infection at 16 months after infection, selection occurred for viruses with deletions in the variable loops VI and V4 in all five animals. The data suggest that these variants evolved as a result of phenotypic selection. To evaluate the biological properties of these variants which have been selected for in the host, we will evaluate whether these viral variants emerged as a result of viral escape from neutralization antibodies, change in receptor usage, or variable viral fitness. We also have a study ongoing in rhesus monkeys to evaluate the quasi-speciation of SIVmac251 following the initiation of infection to determine whether there are particular selection pressures in mucosal compartments that shape the transmitted virus. In a pilot study of 4 rhesus monkeys, we are evaluating virus evolution in mucosal and systemic anatomic compartments to determine whether a local immunologic milieu has an impact on the nature of the mutational changes that the virus accumulates over time. Preliminary data indicate that CTL pressure may be greater on seminal virus than on circulating virus in the first weeks following infection. Although chimeric viruses that express an HIV-1 envelope on an SIV backbone hold promise for facilitating the elucidation of important envelope-associated events in HIV-1 infection and early pathogenesis, the R5-tropic SHIVs that have been generated to date do not cause sufficiently consistent pathogenic consequences to be useful for such studies. To address the need for a consistently pathogenic R5-tropic SHIV, we have constructed SHIV-KB9 viruses that express the V3 loop of two R5-tropic HIV-1 isolates, ADA and YU2. These viruses demonstrate an in vitro tropism for CD4+ T cells that express the CCR5 co-receptor, suggesting that altered tropism of SHFV-KB9 can be achieved by substitution of the gp!20 V3 loop. In a pilot study of the in vivo pathogenic properties of these newly constructed R5-tropic SHIVs, both chimeric constructs replicated to high levels during the period of primary infection and caused a dramatic loss of memory CD4+ T lymphocytes. The most consistently pathogenic of these viruses will be chosen for further development. A cell-free stock will be prepared and titered in vivo for rectal challenge studies to evaluate novel vaccine candidates in monkeys. This chimeric virus will also serve as a backbone for creating a construct to evaluate the pathogenic ramifications of the newly defined signature sequences associated with transmitted HIV-1. Building on previous descriptions of human, mouse, and cynomologous monkey B cell subsets, antibody panels were developed to detect activation, maturation, and function-associated molecules on peripheral blood B cells of rhesus monkeys. To establish baseline data on B cell subsets, peripheral blood from 20 naive rhesus monkeys was evaluated. We then applied these B cell panels to the study of peripheral blood of SlV-infected animals 56 days and 2 years after infection. While the overall number of B cells did not differ significantly different between naive and infected animals, we found clear evidence of pathogenic subset changes, including a loss of naive B cells and an increase in number of activated, memory-type B cells. We are currently extending these studies to include an evaluation lymphatic tissue, including lymph nodes and gastrointestinal tissue, as well as an application of antibodies that detect plasma cell subsets.

先天免疫对早期遏制 HIV-1 复制的贡献仍不清楚。我们有 在一项针对 10 只猴子的研究中，开展了评估 NK 细胞在灵长类慢病毒控制中的作用的研究，其中 NK 通过输注抗 CD16 单克隆抗体，细胞已从动物循环中消除。 在这项正在进行的研究中，我们证明了从细胞中消除 NK 细胞（CD3-、CD20-、NKG2+ 细胞） 猴子外周血连续 7 天不影响血浆病毒 RNA 水平的动力学或损失 SIVmac251 感染后最初几周内的记忆 CD4+ T 淋巴细胞。这一观察 表明 NK 细胞在 SIVmac251 复制的早期控制中发挥的作用可以忽略不计。 观察到 HIV-1 包膜序列在受感染的人类体内随着时间的推移而进化以逃避抗体 中和表明体液免疫反应可能有助于病毒控制，并且在此过程中 对病毒施加选择压力。我们最近优化了抗- CD20 单克隆抗体可消除外周血和淋巴液中的 B 淋巴细胞长达一年 恒河猴的节点。我们正在使用这种抗体作为一项持续的 20 只猴子研究的工具来评估 抗体对早期遏制 SIVmac251 感染的贡献。感染后一百天， 输注抗CD20的猴子的外周血B淋巴细胞仍然没有检测到。血浆病毒RNA 这些动物的水平仍然悬而未决。然而，我们没有观察到显着差异 接受治疗的猴子之间的外周血 CD4+ T 细胞数量和 SIV-gag ELISPOT 反应 实验和对照单克隆抗体输注，表明在此期间病毒复制 实验猴和对照猴的时间段没有显着差异。我们将发起一个 2007 年 5 月进行的一项补充研究，旨在在 SIVmac239 delta nef 疫苗接种的情况下消耗 B 细胞， SIVmac251 挑战评估抗体生成对该活体赋予的保护的贡献 减毒疫苗。 尽管 HIV-1 重复感染的频率尚不清楚，但越来越多的证据表明，个体 已经感染 HIV-1 的人可能会感染异源病毒株。这种临床环境可能 提供了一个重要的窗口，通过该窗口可以观察预防的免疫和病毒学相关性 HIV-1 感染。感染 SIV 的恒河猴模型为研究事件提供了强大的系统 与慢病毒重复感染有关。该模型提供了一种控制潜在变量的方法 可能对传播产生影响，例如两种病毒的遗传相关性、病毒的传播途径和时间 接触情况以及先前感染者的免疫状态。我们创建了一个模型 使用两种 SIV 分离株对猴子进行重复感染，这两种分离株的序列与任何两种循环的 HIV-1 一样不同 隔离。我们还将在这些研究中纳入接受 nef 删除的猴子队列 SIVmac239 病毒，因此先前感染具有复制能力的病毒所提供的保护程度 可以与减毒活病毒疫苗提供的效果进行比较。 我们已经启动了这种慢病毒重复感染的非人灵长类动物模型的研究。为了区分和 为了定量血液中的 SIVmac251 和 SIVsmE660，我们开发了一种定量 PCR 方法 测定血浆样本中两种病毒株的RNA水平。我们建立了两个队列 恒河猴，一只感染了 SIVmac251，另一只感染了 SIVsmE660。我们已经定义了基线 通过表征这些猴子的 CD4+ 和 CD8+ T 淋巴细胞记忆亚群的免疫功能 通过 Elispot 测定和细胞内细胞因子染色观察其 SIV 特异性 T 淋巴细胞功能。这些猴子 然后通过 6 周的直肠内攻击暴露于异源病毒。他们的CD4+ T淋巴细胞 前瞻性评估记忆子集、CD4+ T 淋巴细胞总数和血浆病毒载量。在 完成 6 次直肠内挑战后，这些动物没有证据表明存在重复感染 定量PCR。我们将再监测这些动物四个星期，如果没有证据表明 他们血液中的第二种病毒，我们将使他们面临另一轮 6 种直肠内挑战。如果这些猴子 保持免受重复感染的保护，我们将开始研究以确定介导这种情况的机制 保护。第一项研究将通过以下方式评估 CD8+ T 淋巴细胞在这种保护中的作用： 通过单克隆抗体输注耗尽这些细胞并评估先前提供的保护 感染。 确定传播病毒的性质对于研制有效的 HIV-1 疫苗至关重要。报告由 亨特和同事认为，传播的病毒可能缩短了可变环。阐明 Env 变量环中的删除是否反映了传播瓶颈，我们正在检查病毒 一组 5 只恒河猴的序列进化，这些恒河猴感染了基因定义的种群 SIVmac251通过重复直肠内接种。在病毒复制高峰期和复制高峰期后 6 个月内 感染后，我们没有观察到 env 基因中的可变环缺失。有趣的是，随着这些猴子的进步 感染后16个月从急性感染转为慢性感染，对基因组缺失的病毒进行选择 所有五只动物中都有可变环VI和V4。数据表明，这些变体的进化是由于 表型选择。为了评估这些变体的生物学特性，这些变体已在 宿主，我们将评估这些病毒变体是否是由于病毒中和逃逸而出现的 抗体、受体使用的变化或病毒适应度的变化。 我们还在恒河猴中进行了一项研究，以评估 SIVmac251 的准物种形成 感染的启动以确定粘膜区室中是否存在特定的选择压力 塑造了传播的病毒。在对 4 只恒河猴的初步研究中，我们正在评估病毒在 粘膜和全身解剖区室以确定局部免疫环境是否有影响 关于病毒随时间积累的突变变化的性质。初步数据表明 在感染后的最初几周内，精液病毒的 CTL 压力可能比循环病毒更大。 尽管在 SIV 骨架上表达 HIV-1 包膜的嵌合病毒有望促进 阐明 HIV-1 感染和早期发病机制中重要的包膜相关事件，R5-tropic 迄今为止产生的 SHIV 并没有引起足够一致的致病后果 对于此类研究很有用。为了满足对持续致病性 R5 嗜性 SHIV 的需求，我们构建了 SHIV-KB9 病毒表达两种 R5 向性 HIV-1 分离株 ADA 和 YU2 的 V3 环。这些病毒 证明了表达 CCR5 共受体的 CD4+ T 细胞的体外趋向性，表明改变 SHFV-KB9 的向性可以通过替换 gp!20 V3 环来实现。在一项体内试验研究中 这些新构建的 R5 向性 SHIV 的致病特性，两种嵌合结构都复制到高 初次感染期间的水平，并导致记忆 CD4+ T 淋巴细胞的急剧损失。这 将选择这些病毒中致病性最一致的病毒进行进一步开发。无细胞库存将是 体内制备和滴度用于直肠攻击研究，以评估猴子的新型候选疫苗。这 嵌合病毒还将作为创建构建体的骨干来评估其致病后果 新定义的与传播的 HIV-1 相关的特征序列。 基于之前对人类、小鼠和食蟹猴 B 细胞亚群的描述，抗体组 开发用于检测外周血 B 细胞上的激活、成熟和功能相关分子 恒河猴。建立 20 只恒河猴的 B 细胞亚群、外周血的基线数据 被评价。然后，我们将这些 B 细胞组应用于 SlV 感染动物的外周血研究 56 感染后几天和两年。虽然 B 细胞总数没有显着差异 在幼稚动物和受感染的动物之间，我们发现了致病子集变化的明确证据，包括 幼稚 B 细胞和激活的记忆型 B 细胞数量增加。我们目前正在扩展这些 研究包括评估淋巴组织，包括淋巴结和胃肠组织，以及 检测浆细胞亚群的抗体的应用。