Synthesis of immunogenic glycolipopeptides for the development of vaccines for Ch

描述(申请人提供)：克氏锥虫是一种原生动物寄生虫，在中美洲引起恰加斯病。1100多万人受到感染，每年有多达5万人死于该病慢性阶段的并发症。没有治疗慢性恰加斯病的有效药物，到目前为止也没有疫苗接种。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一种有效的疫苗，为接种疫苗的个人提供全面保护。P.I.S的方法与其他方法有根本的不同，他们试图通过将寄生细胞表面碳水化合物表位整合到完全合成的疫苗中来开发恰加斯病疫苗。锥虫的糖杯是寄生虫的感染形式，含有大量具有末端1-半乳糖基的结构不同的糖链，这是人类所没有的。此外，查格斯病患者的血清含有抗体，称为抗1-Gal，具有独特的功能，即它们通过识别1-半乳糖基表位而导致裂解类鞭毛虫。因此，推测类鞭毛虫的1-半乳糖苷是一种免疫原性极强的B细胞表位，可产生高效的裂解抗体。进一步的假设是，一种完全有效的疫苗应该既能产生裂解抗体，又能产生细胞毒性T细胞。因此，将产生两种包含不同表位的合成疫苗候选并组合使用。我们将合成5个带有末端1-半乳糖苷的小分子低聚糖半抗原，并将其连接到载体蛋白上。这些新糖蛋白将用于1-1，3-半乳糖基转移酶基因敲除(11，3GalT-KO)小鼠的初步筛选免疫，与野生型小鼠不同，这些小鼠对1-半乳糖苷类化合物有反应，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它们裂解类鞭毛虫的能力。然后，能够激发这种免疫反应的1-半乳糖苷表位将被用来通过将它们整合到完全合成的糖脂多肽中来产生潜在的优势疫苗。这些结合物将被设计成包含：(A)一个或多个1-半乳糖苷(S)作为B细胞表位；(B)用于激活辅助T细胞的c4+表位；以及(C)作为一种佐剂的脂类部分，靶向树突状细胞的Toll样受体。另一种类型的结合物将包括(A)脂质；和(B)一个或两个克氏锥虫特异性CD8+T细胞表位，以诱导细胞毒性T细胞。将研究小鼠对这些潜在疫苗的体液和细胞免疫反应。如果成功，这一探索性研究项目将为开发首个有效的恰加斯病人类疫苗创造分子基础，这将对公众健康产生巨大影响，并可能挽救中美洲数千人的生命。此外，所描述的全合成疫苗概念可能对转译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。 公共卫生意义：恰加斯病是中美洲一种威胁生命的疾病，由吸血三联体细菌传播，由单细胞寄生虫克氏锥虫引起，该寄生虫在细胞表面表达某些免疫原性1-半乳糖苷。我们的目标是构建与寄生虫细胞表面相似的全合成糖偶联物，并在小鼠的免疫研究中测试它们。这项研究的结果可能会导致一种对公共健康产生巨大影响的疫苗，不仅可以拯救中美洲数千人的生命，还可以保护美国处于风险之中的个人。

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.