Novel interventions for gammaherpesvirus infection and AIDS-Related Malignancies

为了克服目前疱疹病毒疫苗开发中的挑战，我和我的合作者设计了一种密码子改组的互补基因方法来培养高滴度的复制死亡病毒。这种复制死亡病毒在KSHV共有的关键基因中发生突变，该基因是裂解基因表达所必需的。我们确定两次接种该候选疫苗可产生病毒特异性B和T细胞应答。我们还发现，接种疫苗是针对野生型病毒攻击后的急性病毒复制的保护。在这种初步方法成功的基础上，我们设计了新的重组病毒，这些病毒基因对γ疱疹病毒（包括KSHV）的复制和潜伏期至关重要。这是一种新的疫苗策略，以产生针对结构和非结构基因产物的广泛的B和T细胞库。我们的目标是减少受感染个体的致癌病毒载量和相关癌症负担，特别是HIV/AIDS高危个体。在第二个项目中，CRISPR是我们正在开发的一种基因编辑系统，用于中和它们感染的细胞中的γ疱疹病毒。该系统必须精确地设计以防止对宿主的脱靶损伤，以有效地复制疱疹病毒基因组的多个拷贝。我们通过CRISPR编辑病毒基因组元件成功地减少了鼠γ疱疹病毒的复制。我们接下来应用了一种改进的策略来递送靶向EBV基因组多个位点中的病毒基因和重复元件的指导RNA。下一代测序正在被用于表征所产生的突变类型，我们正在研究这种CRISPR系统如何影响EBV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。

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. 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.