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复制的贡献尚不清楚。我们有 发起研究以评估NK细胞在灵长类动病毒控制中的作用，在10个猴子研究中NK 通过输注抗CD16单克隆抗体，已经从动物的循环中消除了细胞。 在这项正在进行的研究中，我们已经证明了从nk细胞（CD3-，CD20-，NKG2+细胞）消除 猴子的外周血持续7天不影响血浆病毒RNA水平的动力学或丧失 记忆CD4+ T淋巴细胞在感染SIVMAC251后的头几周。这个观察结果 表明NK细胞在SIVMAC251复制的早期控制中起着可忽略的作用。 HIV-1包膜序列随着时间的流逝而在感染人类中逃脱的观察结果 中和表明体液免疫反应可能有助于病毒控制，在此过程中， 对病毒施加选择压力。我们最近优化了抗抗的管理方案 CD20单克隆抗体可从外周血和淋巴消除B淋巴细胞长达一年 恒河猴的节点。我们将这种抗体用作正在进行的20个猴子研究中的工具来评估 抗体对SIVMAC251感染的早期遏制的贡献。感染后一百天， 抗CD20注入的猴子仍然没有可检测到的外围血液B淋巴细胞。血浆病毒RNA 这些动物的水平仍在等待中。但是，我们没有观察到 外周血CD4+ T细胞数量和接收的猴子之间的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 在实验和对照猴子中的时间周期没有显着差异。我们将启动一个 在2007年5月的研究中，在SIVMAC239 DELTA NEF疫苗接种和 SIVMAC251挑战，以评估抗体生成对这种现场保护的保护的贡献 疫苗减弱。 尽管尚不清楚HIV-1超级感染的频率，但有越来越多的证据表明个人 已经感染了HIV-1的病毒可以感染HIV-1。这种临床环境可能 提供一个重要的窗口，通过该窗口可以查看保护的免疫和病毒学相关性 HIV-1感染。 SIV感染的恒河猴模型为研究事件提供了强大的系统 与慢病毒末期感染相关。该模型提供了控制潜在变量的方法 可能会影响传播，例如两种病毒的遗传相关性，病毒的路线和时机 暴露和先前感染的人的免疫状态。我们为 使用两个SIV分离株在猴子中的超级感染，这些SIV分离株在任何两个循环的HIV-1上都一样截然不同 分离物。我们还将包括收到已收到的nef删除的猴子研究同类 SIVMAC239病毒，以便先前感染具有复制能力的病毒的保护程度 可以将活病毒疫苗提供的那些提供的比较。 我们已经在这种非人类的慢病毒末期灵长类动物模型中开始了研究。与众不同 在血液中量化SIVMAC251和SIVSME660，我们开发了一种定量PCR方法 确定血浆样品中两个病毒菌株的RNA水平。我们已经建立了两个队列 恒河猴，一种感染了SIVMAC251，另一种感染了SIVSME660。我们定义了基线 这些猴子的免疫功能通过表征其CD4+和CD8+ T淋巴细胞记忆子集和 通过ELISPOT测定和细胞内细胞因子染色，它们的SIV特异性T淋巴细胞功能。这些猴子 然后通过6个每周直肠内挑战暴露于异源病毒。他们的CD4+ T淋巴细胞 前瞻性评估记忆子集，总CD4+ T淋巴细胞计数和血浆病毒载荷。在 完成6个直肠内挑战的完成，这些动物没有由近次感染的证据 定量PCR。我们将再监视这些动物四个星期，如果没有证据表明 我们的血液中的第二个病毒将使它们面临另一轮直肠内挑战的一轮。如果这些猴子 继续保护免受超级感染的保护，我们将开始研究以确定介导的机制 保护。这些研究中的第一个将是评估CD8+ T淋巴细胞在这种保护中的作用 通过单克隆抗体输注来耗尽这些细胞，并评估先前的保护 感染。 定义传播病毒的性质以创建有效的HIV-1疫苗至关重要。报告 Hunter及其同事建议传播病毒可能缩短了可变环。阐明 env的变量循环中的删除是否反映了传输瓶颈，我们正在检查病毒 序列进化，由5个恒河猴组成的队列，这些猴子被遗传定义的库存感染 SIVMAC251通过重复的直肠内接种。在峰值病毒复制时，最多6个月 感染，我们没有观察到ENV基因中的可变循环缺失。有趣的是，随着这些猴子的发展 从感染后16个月，从急性到慢性感染，对患有缺失的病毒进行了选择 所有五只动物中的可变回路VI和V4。数据表明，这些变体由于 表型选择。评估这些变体的生物学特性 宿主，我们将评估由于中和病毒逃脱而出现的这些病毒变体是否出现 抗体，受体使用变化或病毒适应性变化。 我们还进行了一项在恒河猴中进行的研究，以评估SIVMAC251的准规范 启动感染以确定粘膜隔室中是否有特定的选择压力 那塑造了传播的病毒。在一项针对四只恒河猴的试点研究中，我们正在评估病毒的演变 粘膜和全身解剖室确定局部免疫环境是否有影响 关于病毒随着时间而积累的突变变化的性质。初步数据表明 在感染后的头几周，CTL的CTL压力可能比循环病毒高。 尽管在SIV主链上表达HIV-1信封的嵌合病毒具有促进 在HIV-1感染和早期发病机理中阐明重要的包膜相关事件，R5循环 迄今为止生成的湿因不会引起足够一致的病原后果 对于此类研究有用。为了满足对始终致病的R5诱形SHIV的需求，我们已经构建了 SHIV-KB9病毒表达了两个R5-热带HIV-1分离株ADA和YU2的V3环。这些病毒 证明表达CCR5共受体的CD4+ T细胞的体外向量，表明改变了 可以通过替换GP！20 V3循环来实现SHFV-KB9的向量。在体内的试点研究中 这些新构建的R5-纤维化组织的致病特性，两种嵌合构建体复制到高 原发性感染期间的水平，并导致记忆CD4+ T淋巴细胞的急剧丧失。这 这些病毒的最终致病性将被选择以进一步发展。无单元的库存将是 在体内制备并替代直肠挑战研究，以评估猴子中的新疫苗候选物。这 嵌合病毒还将用作创建构建体以评估病原分析的骨干。 与传输HIV-1相关的新定义的签名序列。 基于对人，小鼠和C猴猴B细胞子集的先前描述，抗体面板 开发以检测外周血B细胞上的激活，成熟和与功能相关的分子 恒河猴。为了建立B细胞子集的基线数据，来自20只幼稚猴子的外周血 被评估。然后，我们将这些B细胞板应用于SLV感染动物的外周血的研究56 感染后2年。虽然B细胞的总数没有显着差异 在幼稚和受感染的动物之间，我们发现了致病子群变化的明确证据，包括丧失 幼稚的B细胞和活化的记忆型B细胞的数量增加。我们目前正在扩展这些 研究包括评估淋巴组织，包括淋巴结和胃肠道组织，以及 检测浆细胞亚群的抗体的应用。