MINIGENE VACCINATION WITH EARLY PRESENTED VIRAL PROTEINSAIDS RELATED RESEARCH

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 目的：研究人类免疫缺陷病毒(HIV)疫苗的新途径。 免疫表位优势导致一个或几个表位组成对SIV感染和疫苗接种的免疫反应的最大部分。我们知道，这些优势表位中的一些在控制SIV方面没有用，因为它们要么快速突变(MAMU-A*01 TAT SL8)，要么根本不能单独控制病毒血症(MAMU-A*01 Gag CM9)。这可能是次显性免疫反应比显性免疫反应更有效，但频率较低。因此，我们设计了一系列Miniges，这是SIV蛋白的区域，编码不超过一个免疫优势表位。通过分离免疫优势表位和亚优势表位，我们希望增加亚优势表位的频率，从而产生更广泛的免疫反应。 此外，虽然我们已经在恒河猴身上成功地进行了以DNA/Ad5为疫苗载体的疫苗试验，但由于以前广泛感染了腺病毒，以及最近使用该载体进行的Merck Step试验结果不佳，腺病毒现在作为疫苗载体在人类中不那么受欢迎。因此，我们在探索微型基因概念的同时，也在探索交替向量的使用。我们将把Miniges放入rBCG、rShigella、Ryf-17D以及可能的其他载体中，接种猕猴，并挑战它们来评估这些新方法的免疫原性。 进度： 继我们之前发表的论文(见新的，逻辑授权)之后，我们开始了一项新的疫苗/挑战研究，目的是增强Gag、Vif和Nef的广度，以及测试新的载体和递送系统。我们刚刚完成了疫苗接种，将于2011年2月开始挑战。 在本研究中，我们将6个Gag基因、2个Vif基因和Nef的核心区分别插入到DNA载体、重组YF-17D载体和Ad5载体中。我们在IAVI的同事对这些微型基因的表达进行了优化。我们使用了几种不同的疫苗策略来比较IL-12在疫苗接种期间的使用，使用Ryf作为增强或启动，以及比较DNA Prime/Ad5 Boost和Ryf Prime/Ad5 Boost。对于DNA引物，我们使用了一种名为DNA电穿孔的新技术，它极大地增强了对DNA质粒中编码序列的免疫反应。在一组中，通过添加编码IL-12的质粒会进一步增强这些反应。我们发现这些免疫反应在很大程度上偏向于CD4+T细胞反应。随后的Ryf和/或rAd5增强导致了更平衡的免疫反应，结果是我们在CD4+和CD8+T细胞亚群中都有强大的免疫反应。我们还使用了Ryf，然后使用rAd5进行了增强，因为同事们已经证明，Ryf的作用更像是质数而不是助推器。对这种方案所产生的反应的比较证实，虽然在Ryf Prime之后观察到的反应很低，但用rAd5增强后的反应比单独用rAd5观察到的反应要大得多。既然这些疫苗接种已经完成，我们将在2011年2月开始挑战动物。 这项研究使用了WNPRC动物服务、遗传学服务和免疫学和病毒学服务。这笔赠款还利用了MHC分型设施以及免疫学和病毒学服务(病毒载量测定、四聚体、流式细胞仪使用、ELISPOTS)的资源。 出版物： Sacha JB，Buechler MB，Newman LP，Reed J，Wallace LT，Loffredo JT，Wilson NA， 沃特金斯警长。猴免疫缺陷病毒特异性CD8+T细胞识别VPR- 和REV衍生的表位在感染后早期。J·维罗尔。2010年10月；84(20)：10907-12。EPub2010年8月4日。PubMed PMID：20686015；PubMed Central。PMCID：PMC2950557。

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 study a new vaccine approach to HIV. Immune epitope dominance results in one or a few epitopes comprising the greatest part of the immune response to SIV infection and vaccination. We know that some of these dominant epitopes are not useful in controlling SIV because they either mutate rapidly (Mamu-A*01 Tat SL8) or simply do not control viremia on their own (Mamu-A*01 Gag CM9). It may be that subdominant immune responses are more efficacious than dominant immune responses, but are at low frequency. Therefore, we have designed a series of Minigenes, which are regions of SIV proteins that encode no more than one immunodominant epitope. By separating immunodominant epitopes from subdominant epitopes, we hope to allow increased frequency of the subdominant epitopes resulting in greater breadth of the immune response. In addition, while we have had successful vaccine trials in rhesus macaques with DNA/Ad5 as vaccine vectors, adenovirus is now less favored as a vaccine vector in humans due to widespread prior infection with adenovirus and due to the poor results obtained in the recent Merck STEP trial using this vector. Therefore, at the same time that we are exploring the minigene concept, we are also exploring the use of alternate vectors. We will put minigenes into rBCG, rShigella, rYF-17D and possibly other vectors, vaccinate macaques and challenge them to assess the immunogenicity of these new approaches. PROGRESS: Following up on our previous publication (see Novel, Logical grant) we have started a new vaccine/challenge study with the goal of enhancing breadth of Gag, Vif and Nef, as well as to test new vectors and delivery systems. We are just finished with the vaccinations and will begin challenging in February 2011. In this study, we inserted six minigenes of Gag, two minigenes of Vif and the core region of Nef into DNA plasmids, recombinant YF-17D vectors and Ad5 vectors. These minigenes were optimized for expression by our colleagues at IAVI. We utilized several different vaccine strategies to compare use if IL-12 during vaccination, use of rYF as either a boost or a prime and comparison of DNA prime/Ad5 boost to rYF prime/Ad5 boost. For the DNA primes, we used a new technique called DNA electroporation, which greatly enhanced immune responses to the sequences encoded in the DNA plasmids. In one group, these responses were further enhanced by the addition of a plasmid encoding IL-12. We found that these immune responses were greatly biased toward CD4+ T cell responses. Subsequent rYF and/or rAd5 boosts resulted in a more balanced immune response, with the result that we have robust immune responses in both CD4+ and CD8+ T cells compartments. We also primed with rYF, then boosted with rAd5, because colleagues have shown that rYF acts more like a prime than a boost. Comparison of the responses engendered by this regimen confirmed that while the responses observed after rYF prime are low, the responses seen after boosting with rAd5 are much greater than that observed with rAd5 alone. Now that these vaccinations are completed, we will start challenging animals in February 2011. This research used WNPRC Animal Services, Genetics Services, and Immunology & Virology Services. This grant also utilized the resources of the MHC typing facility and Immunology and Virology Services (viral load determination, tetramers, flow cytometry instrument utilization, Elispots). PUBLICATION: Sacha JB, Buechler MB, Newman LP, Reed J, Wallace LT, Loffredo JT, Wilson NA, Watkins DI. Simian immunodeficiency virus-specific CD8+ T cells recognize Vpr- and Rev-derived epitopes early after infection. J Virol. 2010 Oct;84(20):10907-12. Epub 2010 Aug 4. PubMed PMID: 20686015; PubMed Central. PMCID: PMC2950557.