Novel interventions for gammaherpesvirus infection and AIDS-Related Malignancies

针对伽马疱疹病毒感染和艾滋病相关恶性肿瘤的新干预措施

• 批准号: 10262616
• 负责人: Laurie T Krug
• 金额: $ 17.33万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262616
• 关键词: AIDS related cancer; AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Acute; B cell repertoire; B-Cell Lymphomas; B-Lymphocytes; Cancer Burden; Cancer Etiology; Cause of Death; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Codon Nucleotides; Complex; Development; Disease; Elements; Engineering; Epithelial Cells; Genes; Genome; Goals; Guide RNA; HIV; Herpesviridae; Herpesvirus Vaccines; Human Herpesvirus 4; Human Herpesvirus 8; Individual; Infection; Intervention; Kaposi Sarcoma; Life; Lymphoma; Lymphoproliferative Disorders; Malignant Neoplasms; Malignant neoplasm of nasopharynx; Methods; Molecular; Morbidity - disease rate; Mus; Mutate; Mutation; Oncogenic; Patients; Pre-Clinical Model; Process; Repetitive Sequence; Research; Risk; Site; Structure; System; T cell response; T-Lymphocyte; Technology; United States; Vaccination; Vaccines; Viral Genes; Viral Genome; Viral Load result; Virus; Virus Replication; antiretroviral therapy; design; gammaherpesvirus; gene complementation; gene product; high risk; lytic gene expression; malignant stomach neoplasm; mortality; next generation sequencing; novel; novel vaccines; pathogen; post-transplant; prevent; primary effusion lymphoma; programs; recombinant virus; success; therapeutic gene; tumorigenesis; vaccine candidate; viral genomics

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.

为了克服当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储备。这种复制死亡病毒在 KSHV 共有的关键基因中发生突变，该基因对于裂解基因表达至关重要。我们确定该候选疫苗的两次给药会产生病毒特异性 B 和 T 细胞反应。我们还发现，疫苗接种可以防止野生型病毒攻击后的急性病毒复制。基于这一初步方法的成功，我们设计了新的重组病毒，可以灭活对伽马疱疹病毒复制和潜伏至关重要的其他病毒基因，包括 KSHV。这是一种新颖的疫苗策略，可产生针对结构和非结构基因产物的广泛 B 和 T 细胞库。我们的目标是减少感染者的致癌病毒载量和相关癌症负担，特别是感染艾滋病毒/艾滋病的高危人群。在第二个项目中，我们正在开发 CRISPR 基因编辑系统，用于中和感染细胞中的伽玛疱疹病毒。该系统必须经过精确设计，以防止对宿主造成脱靶损害，从而有效灭活疱疹病毒基因组的多个副本。我们通过病毒基因组元件的 CRISPR 编辑成功减少了鼠伽马疱疹病毒的复制。接下来，我们应用了一种修改后的策略来提供针对 EBV 基因组多个位点中的病毒基因和重复元件的引导 RNA。下一代测序被用来表征产生的突变类型，我们正在研究该 CRISPR 系统如何影响 EBV 在已知 EBV 感染和转化的多种 B 细胞和上皮细胞中的潜伏期和重新激活。