Eliciting neutralizing antibodies against the HCV E2 envelope glycoprotein

描述（由申请人提供）：大约3%的世界人口感染丙型肝炎病毒（HCV）。作为严重肝脏疾病的主要病因，丙型肝炎病毒是一个紧迫的公共卫生问题。已获许可的抗病毒治疗是非特异性的，目前也没有针对丙肝病毒的疫苗。研究表明，体液反应主要针对E2包膜糖蛋白。为了逃避中和抗体的识别，HCV进化出了糖基化位点，掩盖了E2上的关键功能位点。这些聚糖还可能削弱免疫系统有效诱导针对它们所掩盖的关键表位的中和抗体的能力。我们最近发现，去除HCV E2包膜糖蛋白中的某些糖基化位点会产生一种对HCV阳性患者血清中和高度敏感的病毒。其中两种聚糖被发现掩盖了高度保守的CD81辅助受体结合位点。我们假设用这些E2糖基化突变体免疫小鼠，加上非特异性增强先天免疫的策略，将把体液反应集中在CD81结合位点上，从而引发高滴度的广泛反应性中和抗体。这种抗体在CD81结合位点被阻断的野生型E2诱导时是罕见的。在特异性目的1中，我们将用各种佐剂配制的重组可溶性E2蛋白的糖基化突变体免疫小鼠。由此产生的免疫血清将以效力和中和的广度为特征。在Specific Aim 2中，中和性单克隆抗体（mab）将从具有最高中和性血清滴度的动物中提取。将测试单克隆抗体组合在抑制病毒进入能力方面的协同作用。我们还将确定中和是否与MAb抑制E2-CD81结合相关。在特异性目标3中，我们将确定哪些中和血清、单克隆抗体或单克隆抗体组合可以减弱或阻止病毒逃逸变体的生长。我们的工作将对设计特异性阻断丙型肝炎病毒感染的疫苗具有广泛的意义。公共卫生相关性：丙型肝炎病毒（HCV）是危及生命的肝脏疾病的主要原因。我们寻求设计候选疫苗来特异性阻断HCV感染。

DESCRIPTION (provided by applicant): Approximately 3% of the world population is infected with the Hepatitis C virus (HCV). As a major cause of serious liver disease, HCV represents an urgent public health problem. Licensed antiviral therapies are non-specific and there is currently no vaccine against HCV. It has been shown that the humoral response predominantly target the E2 envelope glycoproteins. In order to escape from recognition by neutralizing antibodies, HCV has evolved glycosylation sites that obscure functionally critical sites on E2. These glycans also probably attenuate the ability of the immune system to effectively elicit neutralizing antibodies against the critical epitopes that they obscure. We recently showed that removal of certain glycosylation sites in the HCV E2 envelope glycoprotein generates a virus that is hyper-sensitive to neutralization by HCV+ patient sera. Two of the glycans were found to mask the highly conserved CD81 coreceptor binding site. We hypothesize that immunizing mice with these E2 glycosylation mutants, coupled with strategies that non-specifically boost innate immunity, will focus the humoral response on the CD81 binding site thereby eliciting high titers of broadly reactive neutralizing antibodies. Such antibodies are rare when elicited by wild type E2 wherein the CD81 binding site is occluded. In Specific Aim 1 we will immunize mice with glycosylation mutants of the recombinant soluble E2 protein formulated in various adjuvants. The resulting immune sera will be characterized for potency and breadth of neutralization. In Specific Aim 2 neutralizing monoclonal antibodies (MAbs) will be derived from animals with the highest neutralizing serum titers. Combinations of MAbs will be tested for synergy in their ability to inhibit viral entry. We will also determine whether neutralization correlates with MAb inhibition of E2-CD81 binding. In Specific Aim 3 we will determine which neutralizing sera, MAbs or combinations of MAbs can attenuate or prevent outgrowth of viral escape variants. Our work will have broad implications for designing vaccines to specifically block HCV infection. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a leading cause of life-threatening liver disease. We seek to design candidate vaccines to specifically block HCV infection.