Entirely Carbohydrate Vaccine Constructs and Their Application in Probing Glycoim

描述(由申请人提供)：该研究计划的主要目标是开发实用的合成方法，用于修饰一种独特的胶囊两性离子多糖(ZPS)、PS A1和结合肿瘤相关碳水化合物抗原(TACA)，以开发完全碳水化合物疫苗构造。PS A1是一种结构特征良好的多糖，含有一个重复的四糖核心，是从脆弱厌氧杆菌NCTC 9343的胶囊多糖复合体(CPC)中分离得到的。已经确定PS A1可以诱使免疫系统通过T和B细胞的参与来诱导细胞和体液免疫功能，从而影响助手和记忆反应事件-这是一个范式转变的发现。目前尚不完全清楚的是，完全基于碳水化合物的构建物作为肿瘤疫苗开发中糖蛋白结合物的替代方法是否具有免疫原性。因此，本研究计划旨在通过三个具体目标来研究和评估创新的疫苗构造：1)开发高效的结合方法来构建结构明确的TACA-PS A1结合疫苗；2)研究TACA-PS A1结合物在小鼠中的免疫学性质，并评价TACA-PS A1增强的抗体对人癌细胞的特异性和细胞毒性；以及3)在体内评价TACA-PS A1激活的免疫治疗肿瘤的有效性。从小鼠免疫中获得的抗血清将用于通过ELISA法测定总抗体效价和抗体亚型，传递有关免疫的信息。此外，还将使用ELISA法来确定针对结合的TACA的抗体的特异性。流式细胞术将用于验证抗体对人类癌细胞株的特异性。利用人类肿瘤细胞系进行补体介导的细胞杀伤(细胞毒)分析，将揭示抗体的功能。最后，将通过使用移植了TA3/Ha小鼠乳腺癌的小鼠模型的活体实验来评估和评估选定的TACA-PS A1构建物治疗癌症的有效性。这项拟议研究的更广泛影响旨在展示完全基于碳水化合物的疫苗如何在免疫系统中引发细胞和体液反应。这项研究计划将有助于改进糖结合化学领域的方法学，更好地理解PS A1作为免疫原在引发免疫反应中的作用，以及评估和评估合理设计的癌症特异性结构。因此，该项目将对癌症治疗产生重要影响，为预防和治疗医学提供一种新的碳水化合物制剂，并增加我们对糖免疫学的理解。我们还希望这一计划能广泛应用于疫苗研究的其他领域。 公共卫生相关性：本申请中提出的研究将提供独特的碳水化合物肿瘤疫苗结构，在预防和治疗医学中具有很高的应用潜力。这项拟议的工作将产生重要影响，因为它将增加我们对碳水化合物如何参与免疫系统的理解。广泛地说，这个项目将通过评估定义明确的半合成碳水化合物结构，从根本上促进我们对体液和细胞糖免疫的了解。

DESCRIPTION (provided by applicant): The principal goal of this research program is to develop practical synthetic approaches for the modification of a unique capsular zwitterionic polysaccharide (ZPS), PS A1, and conjugate tumor-associated carbohydrate antigens (TACAs) to develop entirely carbohydrate vaccine constructs. PS A1 is a structurally well- characterized polysaccharide containing a repeating tetrasaccharide core isolated from the capsular polysaccharide complex (CPC) of the anaerobe Bacterioides fragilis NCTC 9343. It was determined that PS A1 could coax an immune system to elicit cellular and humoral immune functions via T- and B-cell involvement that influenced helper and memorial response events - a paradigm shifting discovery. What is not entirely clear is the immunogenicity of entirely carbohydrate-based constructs as an alternative approach to glycoprotein conjugates for tumor vaccine development. This research program, therefore, aims to study and assess the innovative vaccine constructs using three specific aims: 1) To develop efficient conjugation methods for the construction of structurally well-defined TACA-PS A1 conjugate vaccines, 2) To study the immunological properties of TACA-PS A1 conjugates in mice and evaluate the specificity and cytotoxicity of TACA-PS A1- raised Abs to human cancer cell lines, and 3) To evaluate, in vivo, the efficacy of TACA-PS A1-invoked immunity to treat tumors. Anti-sera, obtained from murine immunizations will be used to determine total antibody (Ab) titers and Ab isotypes by ELISA, relaying information about immunity. Furthermore, ELISA will be used to determine Ab specificity against conjugated TACAs. Flow cytometry will be used to validate Ab specificity with human cancer cell lines. Complement-mediated cell killing (cytotoxicity) analysis using human tumor cell lines, will reveal Ab function. Finally, assessing and evaluating the efficacy of select TACA-PS A1 constructs to treat cancer will be demonstrated through in vivo experiments utilizing murine models grafted with the TA3/Ha murine breast carcinoma. The broader impact of the proposed research aims to demonstrate how entirely carbohydrate-based vaccines elicit cellular and humoral responses in the immune system. This research program will contribute to improved methodologies for the field of glycoconjugation chemistry, a better understanding of the role of PS A1 as an immunogen in eliciting immune responses, and assessment and evaluation of cancer specific constructs that are rationally designed. This project will, therefore, have an important impact on cancer treatment by providing a novel carbohydrate-based agent for preventive and therapeutic medicine and increase our understanding of glycoimmunology. We also expect this program to be broadly applicable to other areas of vaccine research. PUBLIC HEALTH RELEVANCE: The research proposed in this application will provide unique carbohydrate tumor vaccine constructs with high potential for application in preventive and therapeutic medicine. The proposed work will have important impacts as it will increase our understanding of how carbohydrates are involved in the immune system. Broadly, this project will fundamentally advance our knowledge of humoral and cellular glycoimmunity by evaluating well-defined, semi-synthetic carbohydrate constructs.