Present Homologous and Heterologous Antigen with Hepatitis E Virus

描述（由申请人提供）：DNA疫苗的优点包括良好的耐受性、安全性和诱导抗体、细胞毒性T淋巴细胞和T辅助免疫应答的能力，使得其在治疗慢性病毒感染中越来越有吸引力。为了成功地诱导免疫应答，DNA疫苗需要进入靶细胞进行转录和随后的蛋白质产生。理想的递送载体需要安全有效地靶向特定的细胞，在运输过程中提供对DNA质粒的足够保护，以及对任何现有的自身免疫的低应答。缺陷型病毒颗粒，如腺病毒或逆转录病毒，提供了一种有吸引力的选择，其在靶向和保护方面具有很大的益处，但主要缺点是强的自身免疫性以及生产重组病毒颗粒的复杂性。人戊型肝炎病毒非复制型病毒样颗粒（HEV-VLP）是由60个拷贝的重组HEV衣壳蛋白组成的空的二十面体笼。该VLP能够在体外包裹DNA疫苗，并将其递送至胃肠道上皮细胞，诱导抗原特异性的体液和细胞免疫应答。HEV-VLP作为基因载体具有简单的DNA包封过程，但自身免疫的诱导仍是HEV-VLP用于治疗的障碍。 预防针我们对HEV-VLP晶体结构和抗原结构的研究揭示了HEV重组衣壳蛋白在VLP组装和抗原呈递之间相互作用的结构模块性。通过在抗体结合位点插入15个氨基酸，嵌合VLP降低了对抗HEV抗体的反应性，同时保留了二十面体组装。该数据提示我们通过抗体结合位点处的结构改变来降低VLP对抗体诱导的中和的反应性的策略，这是病毒进化以介导其逃避宿主免疫监视的机制。 本申请中提出的实验包括1）使用冷冻电子显微镜和图像重建来鉴定对抗体相互作用至关重要的HEV-VLP表面柔性环：2将短肽插入到鉴定的表面环中以产生对HEV-VLP具有减弱的反应性的嵌合VLP; 3）在递送编码B型肝炎病毒表面抗原的DNA疫苗中评价野生型和嵌合VLP，作为治疗慢性进展型B型肝炎的潜在基因载体。由于HEV是一种肠道传播病毒，因此HEV-VLP能够跨胃肠道粘膜屏障，靶向特定粘膜区域产生抗原。当携带粘膜粘附配体的短肽插入到表面抗原环中时，假设嵌合VLP在靶向粘膜区域中具有强特异性，从而导致粘膜免疫的有效诱导。我们与林杰博士和步行者博士这两位分别在药物发现和乙肝疫苗开发方面的专家合作，期望获得足够的数据，使我们能够重复使用HEV-VLP作为临床基因治疗的载体。

DESCRIPTION (provided by applicant): Advantages of DNA vaccines including well-tolerance, safety and ability to induce antibody, cytotoxic T- lymphocytes, and T-helper immune responses make it more and more attractive in treatment of chronic virus infections. In order to successfully induce immune response, DNA vaccine needs to enter target cell for transcription and subsequent protein production. An ideal delivery carrier needs to be safe and effective in targeting specific cell, provide sufficient protection of DNA plasmid during transportation, as wel as low- responses to any existing self-immunity. Deficient viral particles, such as adenoviruses or retroviruses, offer an attractive option with great benefits in targeting and protection but majr drawbacks on strong self-immunity as well as the complexity in producing recombinant viral particles. Non-replicating virus-like particle derived from human hepatitis E virus (HEV-VLP) is empty icosahedral cage composed of 60 copies of recombinant HEV capsid proteins. This VLP is capable of encapsulating DNA vaccine in vitro, delivering DNA vaccine to epithelium cell at gastrointestinal tract, and inducing antigen-specific humoral and cellular immune responses. The simple procedure in DNA encapsulation makes HEV-VLP of particular interest as gene carrier; however, induction of self-immunity is still the hurdle in using HEV-VLP for therapeutical vaccination. Our studies on HEV-VLP crystal structure and antigenic structure reveal a structural modularity, with which HEV recombinant capsid proteins interplay between VLP assembly and antigen presentation. By carrying insertion of 15 amino acids at an antibody-binding site, the chimeric VLP reduces the reactivity to anti-HEV antibodies meanwhile retains the icosahedral assembly. This data suggests us a strategy to lower the reactivity of VLP to antibody- induced neutralization by structural alteration at antibody-binding sites, a mechanism that viruses have evolved to mediate their escape from host immune surveillance. The proposed experiments in this application include 1) using cryo-electron microscopy and image reconstruction to identify HEV-VLP surface flexible loops that critical to antibody interactions; 2 inserting short peptide into the identified surface loops to create chimeric VLP with attenuate reactivity to HEV-VLP; 3) both wild type and chimeric VLPs will be evaluated in delivery of DNA vaccine encoding the surface antigen of hepatitis B virus, as potential gene carrier for treatment of chronic progressive hepatitis B. Because HEV is an enteric transmitted virus, HEV-VLP is able to transcytose the mucosal barrier at gastrointestinal tract and target specific mucosal region for antigen production. When a short peptide bearing mucosal adhesion ligand is inserted into surface antigenic loops, the chimeric VLP is hypothesized to have strong specificity in targeting mucosal region, leading to potent induction of mucosal immunity. In collaboration with Dr Kit Lam and Dr Christopher Walker, two experts respectively in drug discovery and HBV vaccine development, we expect to obtain sufficient data leading us to repeated use of HEV-VLPs as carrier for clinical gene therapy.