Structure-based HCV vaccine design

全球丙型肝炎病毒感染负担为1.85亿人，每年新增300-400万人 每年都有感染。在美国，由于注射吸毒，年轻人中的丙型肝炎病毒感染正在增加。一个 尽管在开发直接作用抗病毒药物(DAA)方面取得了重大进展，但仍需要预防性疫苗。 用于治疗丙型肝炎病毒感染。累积的证据表明，T和B细胞免疫 有助于控制急性丙型肝炎病毒感染。本申请的重点是基于B细胞的疫苗，该疫苗 已经在技术上具有挑战性，因为病毒包膜E1E2糖蛋白的高度变异性， 保护性抗体的天然靶点。我们已经证明了E2包膜上的免疫原区 丙型肝炎病毒糖蛋白分为五个表位簇，分别为抗原域A-E和高变区 区域1(HVR1)。抗原区A可诱导非病毒中和(非VN)抗体，而HVR1可诱导 分离与病毒逃逸相关的特异性病毒中和(VN)抗体。相比之下，抗原域 B、D和E可诱发VN抗体，这些抗体在主要的丙型肝炎病毒中更广泛地属于VN，并且它们的一些 表位与病毒逃逸无关。基于这些发现，以及最近的证据表明 表位的构象稳定性是免疫原性的关键决定因素，以下问题将是 解决：增加抗原域D和E内VN表位的构象稳定性吗 提高这些保守表位的免疫原性？非VN抗原域A表位可以是 为了降低这些诱饵表位的免疫原性而沉默？我们将使用新的X光 关于E2糖蛋白和与E2 VN结合的人源单抗的结晶学信息 为了解决这些问题，设计了用于免疫学特征的E2变异体。我们的 目标是双重的。首先，我们将稳定抗原结构域E(Aim 1)和D(Aim 2)表位 广泛的病毒中和作用于不同的丙型肝炎病毒基因和亚型分离株，且与 病毒逃脱。第二，我们将下调与非中和相关的抗原域A表位 充当免疫诱骗的抗体(目标3)。结构制导的计算建模将是 用来设计这些改装。携带这些突变的E2变异体将被表征 生化和免疫学，然后评估它们在小鼠体内诱导VN抗体的能力。如果 这些研究的成功将有助于开发一种设计合理的丙型肝炎疫苗，以诱导 广泛的VN抗体可预防不同基因型和亚型的丙型肝炎病毒感染。

The global burden of hepatitis C virus (HCV) infection is at 185 million with an annual rate of 3–4 million new infections each year. In the US, HCV infection is increasing in young adults because of injection drug use. A preventive vaccine is needed in spite of major advances in the development of direct acting antivirals (DAAs) for the treatment of HCV infections. Cumulative evidence has established that both T and B cell immunity contribute to the control of acute HCV infection. The focus of this application is on a B cell-based vaccine that has been technically challenging because of high variability in the viral envelope E1E2 glycoproteins, the natural target of protective antibodies. We have shown that the immunogenic regions on the E2 envelope glycoprotein of HCV segregate into five epitope clusters, designated antigenic domains A–E, and hypervariable region 1 (HVR1). Antigenic domain A elicits non-virus-neutralizing (non-Vn) antibodies and HVR1 elicits isolate-specific virus-neutralizing (Vn) antibodies associated with viral escape. By contrast, antigenic domains B, D, and E elicit Vn antibodies that are more broadly Vn among the major HCV genotypes, and some of their epitopes are not associated with viral escape. Based on these findings, and on recent evidence that the conformational stability of epitopes is a key determinant of immunogenicity, the following questions will be addressed: Does increasing the conformational stability of Vn epitopes within antigenic domains D and E increase the immunogenicity of these conserved epitopes? Can non-Vn antigenic domain A epitopes be silenced in order to decrease the immunogenicity of these decoy epitopes? We will use new X-ray crystallographic information on the E2 glycoprotein, and on human monoclonal antibodies bound to E2 Vn epitopes, to engineer E2 variants for immunological characterization to address these questions. Our objectives are two-fold. First, we will stabilize antigenic domain E (Aim 1) and D (Aim 2) epitopes that mediate broad virus neutralization against diverse HCV genotype and subtype isolates, and that are not associated with viral escape. Second, we will down-modulate antigenic domain A epitopes associated with non-neutralizing antibodies that serve as immunogenic decoys (Aim 3). Structure-guided computational modeling will be employed to design these modifications. E2 variants bearing these mutations will be characterized biochemically and immunologically, followed by evaluation of their capacity to induce Vn antibodies in mice. If successful, these studies will contribute to the development of a rationally designed HCV vaccine to induce broadly Vn antibodies to prevent HCV infection of different genotypes and subtypes.