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 我和我的合作者设计了一种密码子改组的互补基因方法， 复制死亡病毒库存。这种复制死亡病毒在一个关键基因上发生了突变 这是KSHV常见的裂解基因表达所必需的。我们确定两个 该候选疫苗的施用产生病毒特异性B和T细胞应答。我们 还发现接种疫苗对野生型病毒的急性病毒复制具有保护作用， 挑战.在这一初步做法取得成功的基础上，我们设计了新的 复制和潜伏所必需的其他病毒基因的重组病毒 γ疱疹病毒，包括KSHV。这是一种新的疫苗策略， 针对结构和非结构基因产物的B和T细胞库。我们的目标是 为了降低受感染个体中的致癌病毒载量和相关的癌症负担， 尤其是艾滋病高危人群。(2)在第二个项目中，CRISPR是一种 我们正在开发的基因编辑系统，以中和细胞中的γ疱疹病毒， infect.这个系统必须精确设计，以防止对主机造成脱靶损害 以高效地复制疱疹病毒基因组的多个拷贝。我们成功地减少了 通过CRISPR编辑病毒基因组元件的鼠γ疱疹病毒复制。我们接下来 应用一种改良的策略来递送靶向病毒基因和重复序列的引导RNA， 在EBV基因组的多个位点的元件。下一代测序技术被用于 描述产生的突变类型，我们正在研究这种CRISPR系统如何 影响EB病毒在广泛的B细胞和上皮细胞中的潜伏期和再活化， 已知EBV感染和转化。(3)我在分析宿主的免疫反应 HAMB临床试验中患者的外周血单核细胞。我们使用的是流动 流式细胞术检查HIV+和HIV-之间的先天性和适应性免疫反应是否不同 患者我们还研究了免疫反应是否随着化疗而变化， 在临床试验中用于治疗这些患者的免疫疗法。目标是 确定无应答者和应答者的临床应答的免疫相关性， 有效治疗艾滋病相关的恶性肿瘤。

(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.