Entirely Carbohydrate Vaccine Constructs and Their Application in Probing Glycoim

描述（由申请方提供）：本研究项目的主要目标是开发用于修饰独特的荚膜两性离子多糖（ZPS）、PS A1和结合肿瘤相关碳水化合物抗原（TACA）的实用合成方法，以开发完全碳水化合物疫苗构建体。PS A1是一种结构上充分表征的多糖，含有从厌氧菌脆弱拟杆菌NCTC 9343的荚膜多糖复合物（CPC）分离的重复四糖核心。已确定PS A1可以诱导免疫系统通过影响辅助和记忆反应事件的T细胞和B细胞参与来引发细胞和体液免疫功能-一个范式转变的发现。不完全清楚的是完全基于碳水化合物的构建体作为糖蛋白缀合物用于肿瘤疫苗开发的替代方法的免疫原性。因此，本研究计划旨在研究和评估创新疫苗构建体，具有三个特定目标：1）开发用于构建结构明确的TACA-PS A1缀合物疫苗的有效缀合方法，2）研究TACA-PS A1缀合物在小鼠中的免疫学特性，并评估TACA-PS A1- raised Ab对人癌细胞系的特异性和细胞毒性，和3）在体内评价TACA-PS A1引发的免疫治疗肿瘤的功效。将使用从鼠免疫接种中获得的抗血清通过ELISA测定总抗体（Ab）滴度和Ab同种型，传递免疫力相关信息。此外，ELISA将用于确定针对结合TACA的Ab特异性。流式细胞术将用于验证Ab对人癌细胞系的特异性。使用人肿瘤细胞系的补体介导的细胞杀伤（细胞毒性）分析将揭示Ab功能。最后，通过利用移植有TA 3/Ha鼠乳腺癌的鼠模型的体内实验来证明评估和评价选择的TACA-PS A1构建体治疗癌症的功效。拟议研究的更广泛影响旨在证明完全基于碳水化合物的疫苗如何在免疫系统中引起细胞和体液反应。该研究计划将有助于改进糖缀合化学领域的方法，更好地理解PS A1作为免疫原在引发免疫应答中的作用，以及评估和评价合理设计的癌症特异性构建体。因此，该项目将对癌症治疗产生重要影响，为预防和治疗药物提供一种新的基于碳水化合物的药物，并增加我们对糖免疫学的理解。我们还希望该计划能够广泛适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构建体，其在预防和治疗医学中具有很高的应用潜力。这项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。从广义上讲，该项目将从根本上推进我们的体液和细胞糖免疫的知识，通过评估明确的，半合成的碳水化合物结构。

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.