Genetic Modifiers of Immune Evasion by Cytomegalovirus in Glioblastoma

描述(申请人提供)：越来越多的证据表明人类巨细胞病毒(HCMV)与胶质母细胞瘤(GBM)的病因有关：HCMV蛋白被认为是胶质瘤形成的肿瘤促进剂；胶质瘤的分级-以及患者的生存-与HCMV基因产物的水平直接相关。在这项建议中，我们试图了解为什么这种常见的疱疹病毒只在一部分感染者中引起疾病：巨细胞病毒血清阳性率为80%，而基底膜的患病率为0.025%。巨细胞病毒已经进化出高度复杂的免疫逃避策略。一种策略涉及产生两种蛋白质--由基因TRL11/IRL11和UL119-UL118编码--具有Fc？R的功能特性，这可能使病毒通过逃避抗体结合的效应器后果，如ADCC，来逃避宿主的免疫监视。我们实验室最近的研究表明，免疫系统的一个主要基因复合体--14号染色体上三个高度多态的IGHG基因座编码的GM同种异型的等位基因调节着这一病毒策略：编码HCMVTRL11/IRL11的Fc？R与表达GM3+、1-、2-异型的IgG1蛋白的亲和力显著高于表达等位基因GM17+、1+、2+的异型(p=0.0005)。这些观察结果使我们假设GM基因是HCMV-GBM关联的效应修饰物，其潜在机制包括它们对抗HCMV抗体应答的贡献以及它们对病毒免疫逃避策略的调节影响。以下具体目的将验证我们的假设：1)确定GM决定因素在GBM患者中的分布是否与对照组不同。来自GBM患者和对照组的DNA将进行几个GM等位基因的基因分型。由于表达GM3+、1-、2-异型的抗HCMV IgG1抗体与编码的Fc？R有较高的亲和力，其Fc区更容易被清除，从而降低了其通过ADCC和其他Fc介导的效应机制清除病毒的免疫学能力。2)比较GBM患者和对照组的抗巨细胞病毒抗体水平，确定它们是否与特定的GM等位基因有关。用双抗体夹心ELISA法测定患者和对照组血清/血浆中抗人巨细胞病毒糖蛋白B(GB)抗体，并比较两组间的水平。我们还将确定抗体水平是否与特定的GM等位基因相关；3)确定在GBM患者中，HCMV编码的Fc？R蛋白是否与抗HCMV抗体的Fc(GM)区存在差异。我们将克隆并表达编码人巨细胞病毒Fc？RS的胞外区。我们将从GBM患者的血清中提纯针对HCMV GB的抗体。不同GM同种异型的免疫球蛋白分子与HCMV Fc？RS的结合和比较亲和力将通过表面等离子体共振进行监测。拟议中的调查结果可能会为这种每年仅在美国就导致约13000人死亡的恶性肿瘤开辟一条新的调查途径。 公共卫生相关性：胶质母细胞瘤是一种高度致命的脑癌。巨细胞病毒是一种常见的疱疹病毒，与这种恶性肿瘤的病因有关。本项目研究免疫系统的一个主要基因在巨细胞病毒刺激的胶质母细胞瘤的发病机制中的作用。这些研究的结果可能有助于设计针对这种癌症的新的免疫治疗策略。

DESCRIPTION (provided by applicant): Increasing evidence implicates human cytomegalovirus (HCMV) in the etiology of glioblastoma (GBM): HCMV proteins have been implicated as tumor promoters in gliomagenesis; glioma grade-as well as patient survival-directly correlates with the level of HCMV gene products. In this proposal, we seek to understand why this common herpesvirus causes disease in only a subset of those infected: HCMV seroprevalence, 80% vs. the prevalence of GBM, 0.025%. HCMV has evolved highly sophisticated immune evasion strategies. One strategy involves generating two proteins-encoded by genes TRL11/IRL11 and UL119-UL118-that have functional properties of the Fc?R, which may enable the virus to evade host immunosurveillance by evading the effector consequences of antibody binding, such as ADCC. Recent studies from our laboratory show that alleles of a major gene complex of the immune system-GM allotypes encoded by three highly polymorphic IGHG loci on chromosome 14-modulate this viral strategy: The HCMV TRL11/IRL11-encoded Fc?R has significantly higher affinity for IgG1 proteins expressing the GM 3+,1-,2- allotypes than for those expressing the allelic GM 17+,1+,2+ allotypes (p = 0.0005). These observations led us to hypothesize that GM genes are effect modifiers of HCMV-GBM association and the underlying mechanisms include their contribution to anti-HCMV antibody responses and their modulating influence on the viral immune-evasion strategies. The following specific aims will test our hypothesis: 1) Determine if the distribution of GM determinants in GBM patients is different from that in controls. DNA from GBM patients and controls will be genotyped for several GM alleles. Because of their higher affinity to the HCMV-encoded Fc?R, anti-HCMV IgG1 antibodies expressing the GM 3+,1-,2- allotypes would be more likely to have their Fc domains scavenged, thereby reducing their immunological competence to eliminate the virus through ADCC and other Fc-mediated effector mechanisms. Consequently, the frequency of these allotypes would be expected to be higher in patients than in controls; 2) Compare the levels of anti-HCMV antibodies in GBM patients and in controls, and determine if they are associated with particular GM alleles. Antibodies to HCMV glycoprotein B (gB) in the sera/plasma of patients and controls will be quantitated by an ELISA and the levels will be compared between the two groups. We will also determine whether the antibody levels are associated with particular GM alleles; 3) Determine if HCMV-encoded Fc?R proteins bind differentially with genetically disparate Fc (GM) regions of anti-HCMV IgG antibodies in GBM patients. Ectodomains of HCMV-encoded Fc?Rs will be cloned and expressed. We will purify IgG antibodies directed against HCMV gB from the sera of GBM patients. Binding and comparative affinities of IgG molecules of different GM allotypes to the HCMV Fc?Rs will be monitored by surface plasmon resonance. Results from the proposed investigation are likely to open a new avenue of investigation in a malignancy that kills approximately 13000 people every year in the U.S. alone. PUBLIC HEALTH RELEVANCE: Glioblastoma is a highly lethal brain cancer. Cytomegalovirus, a common herpesvirus, has been implicated in the etiology of this malignancy. This project investigates the role of a major gene of the immune system in the etiopathogenesis of cytomegalovirus-spurred glioblastoma. Results from these investigations could help devise novel immunotherapeutic strategies against this cancer.