Novel interventions for gammaherpesvirus infection and AIDS-Related Malignancies

(1)为了克服目前疱疹病毒疫苗开发中的挑战，我和我的同事们设计了一种密码子改组互补基因方法来培育高滴度复制死亡病毒库。这种复制死亡的病毒在KSHV常见的关键基因中发生突变，该基因对裂解基因的表达至关重要。我们确定，该候选疫苗的两次给药会产生病毒特异性的B和T细胞反应。我们还发现，疫苗接种对野生型病毒攻击时的急性病毒复制具有保护作用。在这一初步方法成功的基础上，我们设计了新的重组病毒，可以灭活其他对伽马疱疹病毒的复制和潜伏期至关重要的病毒基因，包括KSHV。这是一种新的疫苗策略，可以产生一系列针对结构和非结构基因产物的B和T细胞。我们的目标是减少感染个人，特别是感染艾滋病毒/艾滋病的高危个人的致癌病毒载量和相关癌症负担。(2)在第二个项目中，CRISPR是我们正在开发的一个基因编辑系统，用于中和感染细胞中的伽马疱疹病毒。该系统的设计必须精准，以防止对宿主的非靶标损害，从而有效地灭活疱疹病毒基因组的多个副本。我们通过CRISPR编辑病毒基因组元件，成功地减少了小鼠伽马疱疹病毒的复制。接下来，我们应用了一种改进的策略来递送针对EBV基因组多个位置中的病毒基因和重复元件的引导RNA。下一代测序正在被用来表征产生的突变的类型，我们正在研究这个CRISPR系统如何影响EBV在已知感染和转化的广泛的B细胞和上皮细胞中的潜伏和重新激活。(3)分析HAMB临床试验患者外周血单个核细胞的宿主免疫反应。我们正在使用流式细胞术来检查HIV+患者和HIV患者之间的先天免疫反应和获得性免疫反应是否不同。我们还研究了临床试验中用于治疗这些患者的化疗和免疫疗法是否会改变免疫反应。目的是为无应答者和应答者识别临床反应的免疫相关性，以找到治疗艾滋病相关恶性肿瘤的有效方法。

(1) To overcome current challenges in the development of herpesvirus vaccines, my collaborators and I devised a codon-shuffled complementing gene method to grow high titer replication-dead virus stocks. This replication-dead virus is mutated in a critical gene common to KSHV that is essential for lytic gene expression. We determined that two administrations of this vaccine candidate generate virus-specific B and T cell responses. We also found that vaccination is protective against acute virus replication upon wild-type virus challenge. Building upon the success of this initial approach, we have designed new recombinant viruses that inactivate other viral genes essential to replication and latency of gammaherpesvirus, including KSHV. This is a novel vaccine strategy to generate a wide repertoire of B and T cells against structural and non-structural gene products. Our goal is to reduce oncogenic viral loads and associated-cancer burdens in infected individuals, especially high-risk individuals with HIV/AIDS. (2) In a second project, CRISPR is a gene-editing system that we are developing to neutralize gammaherpesviruses in the cells they infect. This system must be engineered for precision to prevent off-target damage to the host for efficiency to inactivate multiple copies of herpesvirus genomes. We successfully reduced murine gammaherpesvirus replication by CRISPR editing of viral genomic elements. We next applied a modified strategy to deliver guide RNAs that target viral genes and repetitive elements in the multiple sites of the EBV genome. Next generation sequencing is being used to characterize the types of mutations generated and we are examining how this CRISPR system impacts latency and reactivation of EBV in a broad array of B cells and epithelial cells that EBV is known to infect and transform. (3) I am analyzing the host immune response in the peripheral blood mononuclear cells of patients in HAMB clinical trials. We are using flow cytometry to examine if the innate and adaptive immune response differs between HIV+ and HIV- patients. We also study if the immune response changes with the chemotherapies and immunotherapies that are used to treat these patients in the clinical trials. The goal is to identify immune correlates of the clinical response for non-responders and responders to find effective therapies for AIDS associated malignancies.