Targeted Killing of Herpesvirus-Infected Cells

靶向杀死疱疹病毒感染的细胞

• 批准号: 8555981
• 负责人: Edward Berger
• 金额: $ 21.43万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 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/8555981
• 关键词: Antibodies; Biological Assay; Bos taurus structural-GP protein; Cell Line; Cells; Characteristics; Clinical; Clinical Management; Complement; Diagnosis; Disease; Drug Combinations; Endothelial Cells; Ganciclovir; Genome; Glycoproteins; Goals; Herpesviridae; Human; Human Herpesvirus 8; Immunotoxins; Incidence; Infection; Infusion procedures; Life; Life Expectancy; Lymphoma; Lytic; Lytic Phase; Measurement; Mediating; Multicentric Angiofollicular Lymphoid Hyperplasia; Production; Proteins; Rare Diseases; Reagent; Reporter; Reporting; Research; Specificity; Surface; Surface Antigens; Syndrome; Testing; Viral; Virus; antiretroviral therapy; base; cell killing; cell type; design; immune function; infected B cell; killings; latent infection; research study; tool

Our initial focus was on MCD, a syndrome in which infected B cells are in the lytic phase of the viral replication cycle. Infected cells express several KSHV structural glycoproteins at the surface, including gH/gL, gB, and K8.1A. Though a relatively rare disease, MCD incidence continues to rise despite antiretroviral therapy and the associated improvements in immune function. MCD if a life-threatening disease (median life expectancy <3 years after diagnosis), and no standards for clinical management have been developed. Hence immunotoxins to selectively kill infected B cells may prove beneficial. We previously reported the design and characterization of an immunotoxin, YC15-PE38, that potently killed a gH transfectant cell line and strongly inhibited infectious virus production from Vero-219 cells harboring an episomal infectious KSHV genome with EGFP and RFP that serve as reporters for latent and lytic infection, respectively. We have now characterized a new immunotoxin, 2014-PE38, against a the KSHV glycoprotein K8.1A, which is expressed earlier than gH as cells enter the lytic phase of replication. This immunotoxin potently inhibits infectious virus production from Vero-219 cells; control experiments verified the specificity of this effect based on targeting K8.1A surface antigen, and confirmed that the inhibitory effects were due to selective killing of the virus-producing cells. In combination drug treatment experiments, the immunotoxin complemented the activity of ganciclovir. Assays have been extended to other cell types that are more biologically relevant, incuding the iSLK#10, an endothelial cell line that harbors a reporter-carrying KSHV genome inducible by doxycicline, and BCBL cells derived from a primary infusion lymphoma in which KSHV lytic expression can be experimentally induced. Oddly, neither YC15-PE38 or 2014-PE38 were active against the chronically infected BCBL-1 PEL cell line, or other human PEL cell lines tested, based on both direct cell killing assays and measurements of effects on virus release. Whether this represents an unusual feature of PEL-derived cells, or is characteristic of all KSHV-infected B cell types, remains an important unanswered question.

我们最初的重点是MCD，MCD是一种综合征，其中感染的B细胞处于病毒复制周期的裂解阶段。感染的细胞在表面表达几种KSHV结构糖蛋白，包括GH/GL，GB和K8.1A。尽管是一种相对罕见的疾病，但尽管抗逆转录病毒疗法以及免疫功能的改善，MCD的发病率仍在继续升高。 MCD如果危及生命的疾病（诊断后3年的预期寿命<3年），并且尚未制定临床管理标准。因此，免疫毒素有选择地杀死感染的B细胞可能有益。我们先前报道了免疫毒素YC15-PE38的设计和表征，该免疫毒素有效地杀死了GH转染物细胞系，并从VERO-219细胞中强烈抑制了具有EGFP和RFP的Vero-219细胞的感染性病毒产生，该细胞具有EGFP和RFP，可作为潜在和溶解感染的记者。现在，我们已经针对KSHV糖蛋白K8.1a进行了一种新的免疫毒素，即2014-PE38，它比细胞进入复制的裂解阶段时更早表示。这种免疫毒素可有效抑制VERO-219细胞产生的传染性病毒。对照实验基于靶向K8.1a表面抗原验证了这种作用的特异性，并确认抑制作用是由于选择性杀死病毒的细胞而引起的。在联合药物治疗实验中，免疫毒素补充了甘奇克洛韦的活性。测定已扩展到其他更具生物学相关的细胞类型，即ISLK＃10，这是一种内皮细胞系，该细胞系具有携带记者的KSHV基因组，可通过Doxycicline诱导的KSHV基因组，而BCBL细胞源自BCBL细胞，而BCBL细胞源自kSHV液化淋巴瘤，其中KSHV溶液表达可以实验性地诱导。奇怪的是，基于直接细胞杀伤分析和对病毒释放的影响的测量，YC15-PE38或2014-PE38对慢性感染的BCBL-1 PEL细胞系或其他测试的人类PEL细胞系都没有活性。这是PEL衍生细胞的异常特征，还是所有KSHV感染的B细胞类型的特征，仍然是一个重要的未解决的问题。