Genetic Modifiers of Immune Evasion by Cytomegalovirus in Glioblastoma

描述（由申请人提供）：越来越多的证据表明人巨细胞病毒（HCMV）与胶质母细胞瘤（GBM）的病因有关：HCMV蛋白在胶质瘤形成中作为肿瘤启动子；胶质瘤分级——以及患者生存——与HCMV基因产物的水平直接相关。在本提案中，我们试图理解为什么这种常见的疱疹病毒仅在一部分感染者中引起疾病：HCMV血清阳性率为80%，而GBM的患病率为0.025%。HCMV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，这可能使病毒通过逃避抗体结合的效应后果（如ADCC）来逃避宿主免疫监视。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的等位基因（由14号染色体上三个高度多态性的IGHG基因座编码的gm同种异体）调节了这种病毒策略。R对表达GM 3+、1+、2-等位基因的IgG1蛋白的亲和性显著高于表达GM 17+、1+、2+等位基因的IgG1蛋白（p = 0.0005）。这些观察结果使我们假设转基因基因是HCMV-GBM关联的效应调节剂，其潜在机制包括它们对抗hcmv抗体反应的贡献以及它们对病毒免疫逃避策略的调节作用。以下具体目标将检验我们的假设：1)确定GBM患者中GM决定因子的分布是否与对照组不同。将对GBM患者和对照组的DNA进行几种转基因等位基因的基因分型。因为它们对hcmv编码的Fc？R，表达GM 3+，1-，2-同种异体的抗hcmv IgG1抗体更有可能被Fc结构域清除，从而降低其通过ADCC和其他Fc介导的效应机制消除病毒的免疫能力。因此，这些同种异体的频率预计在患者中高于对照组；2)比较GBM患者和对照组的抗hcmv抗体水平，确定它们是否与特定的GM等位基因相关。采用ELISA定量检测患者和对照组血清/血浆中HCMV糖蛋白B （gB）抗体，并比较两组之间的水平。我们还将确定抗体水平是否与特定的转基因等位基因相关；3)确定是否hcmv编码Fc？在GBM患者中，R蛋白与抗hcmv IgG抗体基因不同的Fc （GM）区域结合的差异。hcmv编码Fc？Rs将被克隆和表达。我们将从GBM患者的血清中纯化针对HCMV gB的IgG抗体。不同GM异型IgG分子与HCMV Fc的结合及比较亲和力Rs将由表面等离子体共振监测。拟议的调查结果可能会为恶性肿瘤的调查开辟一条新的途径，每年仅在美国就有大约13000人死亡。

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.