Antigen-specific T-cell activation, application to vacci

抗原特异性T细胞激活，在疫苗中的应用

• 批准号: 6946729
• 负责人: JAY A BERZOFSKY
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6946729
• 关键词: AIDS therapy; AIDS vaccines; HIV envelope protein; HIV infections; MHC class I antigen; Macaca mulatta; T lymphocyte; biotechnology; cellular immunity; clinical trial phase I; colony stimulating factor; cytokine; epitope mapping; genetically modified animals; hepatitis C virus; human subject; human therapy evaluation; immunoregulation; laboratory mouse; leukocyte activation /transformation; liver neoplasms; neoplasm /cancer vaccine; neutralizing antibody; patient oriented research; protein engineering; synthetic vaccines; tissue /cell culture; vaccine development; vector vaccine

We studied mechanisms for T cell recognition of antigens in association with major histocompatibility complex (MHC)-encoded molecules, and applications to the design of synthetic vaccines for AIDS and cancer. We have been characterizing the helper and cytotoxic T lymphocyte (CTL) responses to HIV envelope and reverse transcriptase, mapping the key epitopes, and defining the role of individual residues in these epitopes to be able to modify the structures to make more potent immunogens as vaccines. We have made vaccine constructs in which clusters of helper epitopes are synthesized coupled to a peptide that is a CTL epitope presented promiscuously by multiple class I MHC molecules in the human and mouse as well as a neutralizing antibody epitope. These constructs can induce all three arms of the immune response, neutralizing antibodies, CTL, and Th1 helper cells. Results of the first arm of a phase I clinical trial with one of these peptides show ability to induce CTL, helper T cell responses, and neutralizing antibodies to HIV in at least a subset of human recipients. Currently, we are developing new approaches in mouse models to develop second generation vaccine constructs. We have shown proof of principle that we can modify the sequence of a helper epitope of HIV to make it more immunogenic and also much more potent, when coupled to a CTL epitope, in eliciting CTL and protecting against viral infection. The enhanced helper epitopes elicit a stronger Th1 response and upregulate CD40L on the helper cells, which in turn induce more IL-12 production by dendritic cells, which then polarize the T helper cells to Th1. We are applying this "epitope enhancement" approach to conserved HIV helper and CTL epitopes from env, gag, and pol, presented by human class II and class I HLA molecules, as well as to hepatitis C virus (HCV) epitopes presented by human HLA-A2.1 (see below). We have developed an enhanced HIV reverse transcriptase epitope which is the subject of a clnical trial to be carried out with Dr. Robert Yarchoan, HAMB, CCR, NCI, and we have characterized human responses to an envelope helper epitope, which we have also enhanced. We have discovered ways of increasing CTL, helper, and antibody responses and steering them toward desired phenotypes, such as Th1 or Th2 or particular antibody isotypes, by incorporating cytokines into the emulsion adjuvant with the antigen. We compared a panel of 8 cytokines for their effects on 8 types of immune response, and discovered a novel synergy between GM-CSF and IL-12 and between TNF and IL-12 in induction of CTL. We found that all 3 cytokines provide triple synergy for induction of CTL with a peptide vaccine, for induction of interferon-gamma, and for protection against viral challenge in vivo, which we show to be interferon-gamma dependent. The mechanism of this synergy appears to relate to the upregulation of antigen presenting function by GM-CSF and of the IL-12 receptor by TNF and IL-12. Further, we have recently shown that GM-CSF that recruits dendritic cells and CD40L that matures them will synergize as a potent vaccine adjuvant combination for induction of CTL and protection against viral infection. We have even further enhanced this combination by a push-pull approach in which this potent combination of cytokine and costimulatory molecule is complemented by blocking a suppressive pathway with an inhibitor of IL-13, optimizing the vaccine-induced CTL response and protection. We have shown that high avidity CTL specific for HIV-1 envelope peptide are much more effective at clearing a recombinant vaccinia virus expressing HIV gp160 from SCID mice than are low avidity CTL specific for the same peptide-MHC complex, and have worked out two complementary mechanisms involving the ability of high avidity CTL to kill cells earlier in virus infection before viral progeny are produced, and to lyse targets more quickly. We have finally developed a method to preferentially elicit higher avidity CTL with a vaccine, by using costimulatory molecules to allow a response at lower antigen dose. However, we found that high avidity CTL are exquisitely sensitive to high dose antigen and will undergo programmed cell death, mediated by TNF and the TNF receptor II, but also requiring a permissive state involving a decrease in Bcl-2, IAP1, and TRAF2, and correlating with downmodulation of the T cell receptor. This effect may explain clonal exhaustion in viral infections. We have also found that IL-15 incorporated in a vaccine induces CTL of a different character that are longer-lived memory cells, with higher levels of IL-15Ra, greater responsiveness to IL-15 in vitro and greater homeostatic proliferation in vivo, and higher avidity for antigen. We have shown for the first time that protection against mucosal transmission of virus can be mediated by CD8 CTL without antibodies, but requires that the CTL be present at the mucosal site of transmission, whereas systemic CTL are not sufficient. The protection can be accomplished by intrarectal immunization with a peptide vaccine and increased by inclusion of IL-12 and GM-CSF with the vaccine. We found that endogenous IL-12 is less inhibited by the mucosal adjuvant LT(R192G) than by cholera toxin, and substituting this, the mucosal CTL response and protection are less dependent on exogenous IL-12. Using this mutant LT, we immunized MamuA*01-positive Rhesus macaques intrarectally with a similar peptide AIDS vaccine and induced CTL in the colon and mesenteric lymph nodes that have impacted the clearance of virus after intrarectal challenge with pathogenic AIDS virus SHIV-Ku. Intrarectal immunization was more effective than subcutaneous immunization with the same peptide vaccine at protecting against SHIV, in part because we found the induction of mucosal CTL provided for greater clearance from a major site of virus replication, the gut mucosa, which was seeding the bloodstream. We have commenced a second Rhesus macaque mucosal vaccine study using mucosal peptide priming, with a combination of cytokines as adjuvants, and using mucosal boosting with a recombinant poxvirus, to prevent AIDS virus transmission across a mucosal barrier. The peptide prime-NYVAC viral vector boost strategy appears more effective than either component alone in inducing both CTL responses and control of viremia after challenge. With regard to cancer, we identified several CTL epitopes in proteins of hepatitis C virus (HCV), that causes liver cancer, using a novel approach, and have analyzed the role of each amino acid residue in order to modify one of the peptides to make a more potent vaccine. Using this epitope enhancement approach, we could increase the immunogenicity of an epitope of the HCV core protein, presented by the most common human class I HLA molecule, HLA-A2.1, both for HLA-A2.1-transgenic mice in vivo and for human T cells in vitro. This enhanced epitope is being incorporated into a vaccine. We are attempting to enhance other HCV core epitopes to incorporate into a DNA vaccine. We also developed a model of immunosurveillance of cancer in which tumors are rejected by CD8 T cells, but the rejection is incomplete in the presence of normal CD4 regulatory cells, and an escape variant of the tumor recurs. However, depletion of CD4 cells allows complete eradication of the tumor by CD8 cells. Using receptor knock-out mice, we found that the key regulatory cytokine inhibiting immunosurveillance against cancer was IL-13, acting through the IL-4 receptor/STAT6 pathway, although IL-4 itself was neither necessary nor sufficient. We discovered that the major source of IL-13 was NKT cells, and that absence of these in CD1-knockout mice prevented tumor recurrence in these mice.

我们研究了与主要组织相容性复合物（MHC）编码分子相关的T细胞识别的机制，并应用于艾滋病和癌症的合成疫苗设计。我们一直在表征助手和细胞毒性T淋巴细胞（CTL）对HIV包膜和逆转录酶的反应，映射关键表位，并确定各个残基在这些表位中的作用，以便能够修饰结构以使疫苗成为更多有效的免疫原。我们制造了疫苗构建体，其中将辅助表位的簇簇合成与肽偶联，该肽是人类和小鼠中多个I类MHC分子以及中和抗体表位的CTL表位。这些构建体可以诱导免疫反应的所有三个臂，中和抗体，CTL和Th1辅助细胞。 I期临床试验的第一臂的结果，其中一项肽表明在至少一部分人类受体中诱导CTL，辅助T细胞反应以及对HIV的中和抗体的能力。当前，我们正在开发鼠标模型中的新方法，以开发第二代疫苗构建体。我们已经显示了原理证明，即我们可以修改HIV的辅助辅助表位的序列，从而使其更加免疫原性，并且在耦合到CTL表位时，可以引起CTL并保护病毒感染。增强的辅助辅助表位会引起更强的Th1响应，并在辅助细胞上上调CD40L，进而诱导树突状细胞的更多IL-12产生，然后将T辅助细胞偏振至TH1。我们正在将这种“表位增强”方法应用于ENV，GAG和POL的保守HIV助手和CTL表位，由人类II类和I类HLA分子提出，以及乙型肝炎病毒（HCV）表位，由人类HLA-A2.1提出。我们已经开发了一种增强的HIV逆转录酶表位，这是与Robert Yarchoan博士，汉堡，CCR，NCI进行的clnical试验的主题，我们已经表征了人类对信封助理表位的反应，我们也已经增强了人类的辅助辅助表位。我们发现了增加CTL，助手和抗体反应的方法，并将其转向所需的表型，例如Th1或Th2或特定的抗体同型，或通过将细胞因子掺入与抗原的乳液辅助剂中。我们比较了一组8个细胞因子对8种免疫反应的影响，并发现了GM-CSF和IL-12之间的新型协同作用以及TNF和IL-12之间的CTL诱导。我们发现，所有3种细胞因子都提供了三重协同作用，可用于用肽疫苗诱导CTL，用于诱导干扰素 - 伽玛以及在体内防止病毒攻击的保护，我们表明这是依赖性的γ-γ。该协同作用的机制似乎与GM-CSF和TNF和IL-12受IL-12受体的抗原功能的上调有关。此外，我们最近表明，募集它们的树突状细胞和CD40L的GM-CSF将作为有效的疫苗辅助组合协同作用，以诱导CTL和防止病毒感染。我们通过推pull方法进一步增强了这种组合，在这种方法中，通过用IL-13的抑制剂阻止抑制性途径，可以互补细胞因子和共刺激分子的有效组合，从而优化了疫苗诱导的CTL响应和保护。我们已经表明，与HIV-1包膜肽相比，对HIV-1包膜肽特异性的高流相关CTL在清除SCID小鼠中表达HIV HIV GP160的重组疫苗的病毒更有效，而不是针对同一肽-MHC复合物特异性的低ctl，并且已经杀死了较高的互补机制，以前杀死了两个互补机制。目标更快。我们最终开发了一种通过使用costimulation分子允许在较低抗原剂量下的反应，优先通过疫苗引起较高的疫苗CTL的方法。但是，我们发现高剂量抗原的高潮CTL非常敏感，并将经历由TNF和TNF受体II介导的程序性细胞死亡，但也需要允许的状态，涉及Bcl-2，IAP1和TRAF2的降低，并与T细胞受体下降相关。这种作用可能解释了病毒感染的克隆疲劳。我们还发现，在疫苗中掺入IL-15会诱导具有较长寿命的记忆细胞的CTL，具有较高水平的IL-15RA，对IL-15的体外反应性更高，体内稳态增殖较大，并且对抗原的稳态增殖更高。 我们首次表明，CD8 CTL无抗体可以介导防止病毒的粘膜传播，但要求CTL存在于传播的粘膜部位，而全身CTL则不足。可以通过肽疫苗内直肠免疫来实现该保护，并通过将IL-12和GM-CSF纳入疫苗中增加。我们发现，内源性IL-12受粘膜辅助LT（R192G）的抑制作用要小于霍乱毒素，而取代此症状，粘膜CTL反应和保护较少依赖于外源IL-12。使用该突变体LT，我们对MAMUA*01阳性恒河猕猴在结肠和肠系膜淋巴结中与类似的肽AIDS疫苗内部内部直立，并诱导CTL，这些淋巴结影响了病毒在与致病性AIDS Virus Shiv-shiv-Ku的直肠挑战后对病毒清除的清除。直肠内免疫比皮下免疫更有效，在预防SHIV时具有相同的肽疫苗，部分原因是我们发现诱导粘膜CTL可从病毒复制的主要部位，肠粘膜的主要清除，这是肠粘膜，这是在血液中播种。我们使用粘液肽启动进行了第二次恒河猴粘膜疫苗研究，并结合了细胞因子作为佐剂的组合，并与重组痘病毒一起使用粘膜促进，以防止AIDS病毒在粘膜屏障中的传播。肽Prime-NYVAC病毒载体增强策略似乎比单独的组件在诱导CTL反应和挑战后的病毒血症控制方面更有效。 关于癌症，我们确定了丙型肝炎病毒（HCV）中引起肝癌的几个CTL表位，它们使用一种新方法，并分析了每个氨基酸残基的作用，以修改其中一种肽以制造更有效的疫苗。使用这种表位增强方法，我们可以提高由最常见的人类I类HLA分子HLA-A2.1提出的HCV核心蛋白表位的免疫原性，均用于HLA-A2.1- TRANSGENIC小鼠体内的HLA-A2.1-转基因小鼠。这种增强的表位被掺入疫苗中。我们正在尝试增强其他HCV核心表位以掺入DNA疫苗中。我们还开发了一种癌症免疫监视的模型，其中肿瘤被CD8 T细胞拒绝，但是在存在正常CD4调节细胞的情况下，排斥反应是不完整的，并且肿瘤的逃脱变体又出现了。但是，CD4细胞的耗竭允许CD8细胞完全消除肿瘤。使用受体敲除小鼠，我们发现抑制癌症免疫监测的关键调节细胞因子是IL-13，它通过IL-4受体/STAT6途径作用，尽管IL-4本身既不必需也​​不足够。我们发现IL-13的主要来源是NKT细胞，并且在CD1-敲除小鼠中缺乏这些细胞会阻止这些小鼠的肿瘤复发。