Synthesis of immunogenic glycolipopeptides for the development of vaccines for Ch

描述（由申请人提供）：克氏锥虫是一种在中美洲引起恰加斯病的原生动物寄生虫。超过1100万人受到感染，每年有多达5万人死于该病慢性阶段的并发症。目前还没有治疗慢性恰加斯病的有效药物，也没有疫苗接种。这一试点项目的长期目标是获得关键的探索性疫苗/免疫反应数据，这些数据最终将导致研制出一种有效的疫苗，为接种疫苗的个人提供充分保护。侦探他的方法与其他人开发恰加斯病疫苗的尝试有着根本的不同，他们的想法是将寄生细胞表面碳水化合物表位纳入完全合成的疫苗中。锥马鞭毛虫的糖杯是寄生虫的感染形式，含有大量具有末端1-半乳糖基的结构异质聚糖，这些聚糖对人类来说是外来的。此外，Chagasic患者的血清中含有抗体，称为抗1-gal，具有独特的功能，即它们通过识别1-半乳糖表位导致锥乳糜虫的裂解。因此，我们假设锥乳虫的1-半乳糖苷是极具免疫原性的b细胞表位，可引发高效的裂解抗体。进一步的假设是，一个完全有效的疫苗应该同时引发溶性抗体和细胞毒性t细胞。因此，将产生两种含有不同表位的合成候选疫苗，并将其联合使用。将合成5个末端含有1-半乳糖苷的小合成寡糖半抗原并与载体蛋白结合。这些新糖蛋白将用于1-1,3-半乳糖转移酶敲除（11,3 galt - ko）小鼠的初始筛选免疫，与野生型小鼠相比，这些小鼠对1-半乳糖苷有反应，因此是一个很好的人类模型。免疫动物的血清将在体外测试其溶锥乳线虫的能力。能够引起这种免疫反应的1-半乳糖苷表位随后将被用于通过将其整合到完全合成的糖多糖肽中来产生潜在的优质疫苗。这些偶联物将被设计成含有：(a)一个或多个1-半乳糖苷(s)作为b细胞表位；(b)活化辅助性t细胞的CD4+表位；(c)脂质部分作为佐剂，靶向树突状细胞的toll样受体。另一种缀合物由(a)脂质组成；(b)一个或两个克氏锥虫特异性CD8+ t细胞表位诱导细胞毒性t细胞。小鼠对这些潜在疫苗的体液和细胞免疫反应将被研究。如果取得成功，这一探索性研究项目将为研制首个防治恰加斯病的有效人体疫苗奠定分子基础，这将对公共卫生产生巨大影响，并可能挽救中美洲成千上万人的生命。此外，所述的完全合成疫苗概念可能对转化研究具有更广泛的相关性，因为它可能适用于其他地方性寄生虫病和传染病。

DESCRIPTION (provided by applicant): Trypanosoma cruzi is a protozoan parasite that causes Chagas disease in Central America. Over 11 million people are infected, and up to 50,000 die each year due to complications during the chronic stage of the disease. No effective drugs exist to treat chronic Chagas disease, and no vaccination is available to date. The long-term goal of this pilot project is to obtain critical exploratory vaccine/immune response data that will ultimately lead to an effective vaccine that provides full protection to immunized individuals. The P.I.'s approach differs fundamentally from others' attempts to develop a vaccine for Chagas disease by the idea of incorporating parasitic cell surface carbohydrate epitopes into fully synthetic vaccines. The glycocalix of trypomastigotes, the infective forms of the parasite, contains large amounts of structurally heterogeneous glycans with terminal 1-galactosyl moieties, which are foreign to humans. Furthermore, the sera of Chagasic patients contain antibodies, known as anti-1-Gal, with a unique function, i.e. they cause lysis of trypomastigotes by recognition of 1-galactosyl epitopes. Therefore, it is hypothesized that the trypomastigotes' 1-galactosides are extremely immunogenic B-cell epitopes that elicit highly effective lytic antibodies. It is further hypothesized that a fully effective vaccine should elicit both, lytic antibodies as well as cytotoxic T-cells. Therefore, two types of synthetic vaccine candidates that contain different epitopes will be generated and used in combination. Five small synthetic oligosaccharide haptens with terminal 1-galactosides will be synthesized and conjugated to carrier proteins. The neoglycoproteins will be used for initial screening immunization of 1-1,3- galactosyltransferase knockout (11,3GalT-KO) mice, which in contrast to wild-type counterparts, are responsive to 1-galactosides, and are therefore a good model for humans. The sera of immunized animals will be tested for their ability to lyse trypomastigotes in vitro. The 1-galactoside epitopes that are capable of evoking such an immune response will then be used to generate potentially superior vaccines by incorporating them into fully synthetic glycolipopeptides. These conjugates will be designed to contain: (a) one or more 1- galactoside(s) as B-cell epitopes; (b) a CD4+ epitope for the activation of helper T-cells; and (c) a lipid moiety as an adjuvant that targets Toll-like receptors of dendritic cells. Another type of conjugate will consist of (a) a lipid; and (b) one or two Trypanosoma cruzi specific CD8+ T-cell epitopes to elicit cytotoxic T-cells. The humoral and cellular immune responses of mice to these potential vaccines will be studied. If successful, this exploratory research project will create the molecular basis for the development of the first effective human vaccine for Chagas disease, which would have a tremendous impact on public health, and may save thousands of lives in Central America. In addition, the described concept of fully synthetic vaccines may have a much broader relevance for translational research, as it may be applicable to other endemic parasitic and infectious diseases. PUBLIC HEALTH RELEVANCE: Chagas disease is a life-threatening disease in Central America that is transmitted by the blood- sucking triatome bug, and is caused by a unicellular parasite, Trypanosoma cruzi, that expresses certain immunogenic 1-galactosides on its cell surface. Our goal is to construct fully synthetic glycoconjugates that resemble the cell surface of the parasite, and test them in immunization studies in mice. The results of this research could lead to a vaccine which would have a tremendous impact on public health, not only by saving thousands of lives in Central America, but also by protecting individuals under risk in the U.S.