VACCINE REGIMENS TO INDUCE CD4+ AND CD8+ T CELLS AGAINST SIV EPITOPES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 目的：比较6种新疫苗方案诱导的CD 4+和CD 8 + T细胞对SIV抗原表位的免疫应答。 最近临床艾滋病毒疫苗的失败强调了更彻底地评估艾滋病毒基础科学以及测试新疫苗方案和载体的重要性。为了克服更传统的基于载体的疫苗的局限性，我们的实验室开发了几种新的免疫策略。这些新方法将使我们能够直接引发特异性T细胞反应，这是以前其他疫苗载体和方案不可能实现的。这将使我们能够分析特异性T细胞应答在控制SIV复制中的作用-亚显性CD 8 + T细胞和病毒特异性CD 4 + T细胞的作用。 在该资助的R21阶段，我们将比较六种新型疫苗接种方案：肽脉冲树突状细胞、肽缀合纳米珠、肽脉冲PBMC、与乙型肝炎B核心抗原（HBcAg）载体基因融合的SIV肽、电穿孔DNA+IL-12和腺病毒5。 进展： 我们已完成R21阶段第1组和第2组动物的接种。这些动物接受用三个Mamu-DRbw*606限制性CD 4表位脉冲的自体树突状细胞，并用用相同表位脉冲的自体PBMC或肽缀合的纳米珠引发。最终加强后两周，没有动物产生任何可检测的表位特异性CD 4 + T细胞应答，如通过ELISPOT或ICS检测的。 HBcAg载体由Deborah Fuller博士完成，每个选定的Mamu-A*01限制性CD 8表位插入到单个载体中。6只Mamu-A*01+动物（第3组和第4组）接受5剂HBcAg载体。第5次接种后，没有动物产生任何可检测的表位特异性CD 8 + T细胞应答;然而，它们对Fuller博士提供的肝炎肽应答良好，表明它们确实接受了疫苗接种。在最后一次初免后一个月，一组接受用每个A*01表位脉冲的自体PBMC，而另一组接受与纳米珠缀合的A*01表位。在加强这些动物后，我们仍然没有看到可检测到的表位特异性CD 8 + T细胞应答。 另外，我们制备了含有三个CD 4表位和五个CD 8表位的DNA和腺病毒载体。我们用这些构建体接种动物，希望引发表位特异性CD 4+和CD 8 + T细胞应答。在Ad 5加强免疫两周后，我们检测到针对疫苗中所有三个表位的高频率SIV特异性CD 4 + T细胞应答。三只动物中的两只对所有三个表位做出反应，而第三只动物对三个表位中的两个做出反应。接受CD 8表位的所有三只动物对Env：Env TL 9中的一个表位产生高频应答。一只动物产生额外的Pol LV 10特异性CD 8 + T细胞应答。虽然表位特异性CD 4疫苗接种是成功的，但所测试的疫苗方案均未引发几种高频率表位特异性CD 8 + T细胞应答。 正在编写一份出版物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Objective: To compare six novel vaccine regimens to induce CD4+ and CD8+ T Cells against SIV epitopes. Recent failures in clinical HIV vaccines have underscored the importance of more thoroughly evaluating basic science of HIV as well as testing new vaccine regimens and vectors. In an effort to overcome the limitations of more traditional vector-based vaccines, our laboratory has developed several novel immunization strategies. These new methods will allow us to directly prime specific T cell responses in a manner that previously has been impossible with other vaccine vectors and regimens. This should allow us to dissect the contributions of specific T cell responses in the control of SIV replication -- for the roles of both subdominant CD8+ T cells and virus-specific CD4+ T cells. In the R21 phase of this grant, we will compare six novel vaccination regimens: peptide-pulsed dendritic cells, peptide-conjugated nanobeads, peptide-pulsed PBMC, SIV peptides fused to a Hepatitis B core antigen (HBcAg) carrier gene, electroporated DNA+IL-12 and Adenovirus5. PROGRESS: We have completed vaccinating animals in groups one and two of the R21 phase. These animals received autologous dendritic cells pulsed with three Mamu-DRbw*606-restricted CD4 epitopes and primed with either autologous PBMC pulsed with the same epitopes or peptide-conjugated nanobeads. Two weeks after the final boosts, no animals made any detectable epitope-specific CD4+ T cell responses as detected by ELISPOT or ICS. The HBcAg vectors were completed by Dr. Deborah Fuller with each of the selected Mamu-A*01-restricted CD8 epitopes inserted into individual vectors. Six Mamu-A*01+ animals, groups three and four, received five doses of the HBcAg vector. After the fifth dose, no animals made any detectable epitope-specific CD8+ T cell response; however, they did respond well to Hepatitis peptide provided by Dr. Fuller indicating that they did receive doses of the vaccine. One month after the final prime, one group received autologous PBMC pulsed with each of the A*01 epitopes, while the other group received the A*01 epitopes conjugated to nanobeads. After boosting these animals, we still saw no detectable epitope-specific CD8+ T cell responses. Additionally, we made DNA and Adenovirus5 vectors containing the three CD4 epitopes and the five CD8 epitopes. We vaccinated animals with these constructs in the hope of eliciting epitope-specific CD4+ and CD8+ T cell responses. Two weeks after the Ad5 boost, we detected high frequency SIV-specific CD4+ T cell responses against all three epitopes in the vaccine. Two of the three animals made responses to all three epitopes, while the third animal made responses to two of the three epitopes. All three animals that received the CD8 epitopes made a high frequency response to one epitope in Env: Env TL9. One animal made an additional Pol LV10-specific CD8+ T cell response. While the epitope-specific CD4 vaccination was successful, none of the tested vaccine regimens elicited several high frequency epitope-specific CD8+ T cell responses. A publication is in preparation.