Entirely Carbohydrate Vaccine Constructs and Their Application in Probing Glycoim

描述（由申请人提供）：本研究项目的主要目标是开发一种实用的合成方法，用于修饰一种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（TACAs），以开发完全碳水化合物的疫苗结构。PS A1是从厌氧菌脆弱杆菌NCTC 9343的荚膜多糖复合体（CPC）中分离出来的重复四糖核心，是一种结构表征良好的多糖。我们确定，PS A1可以通过T细胞和b细胞的参与诱导免疫系统引发细胞和体液免疫功能，从而影响辅助和记忆反应事件，这是一个范式转变的发现。目前尚不完全清楚的是，完全以碳水化合物为基础的构建物作为糖蛋白偶联物的替代方法用于肿瘤疫苗开发的免疫原性。因此，本研究计划旨在通过以下三个具体目标来研究和评估创新疫苗结构：1)建立高效的偶联方法，构建结构明确的TACA-PS A1偶联疫苗；2)研究TACA-PS A1偶联物在小鼠体内的免疫学特性，评价TACA-PS A1上调抗体对人癌细胞的特异性和细胞毒性；3)在体内评价TACA-PS A1激活免疫治疗肿瘤的效果。从小鼠免疫中获得的抗血清将用于ELISA测定总抗体（Ab）滴度和Ab同种型，传递免疫信息。此外，ELISA将用于测定Ab对偶联TACAs的特异性。流式细胞术将用于验证Ab对人类癌细胞系的特异性。利用人肿瘤细胞系进行补体介导的细胞杀伤（细胞毒性）分析，将揭示Ab的功能。最后，我们将通过植入TA3/Ha小鼠乳腺癌的小鼠模型进行体内实验，来评估和评价所选择的TACA-PS A1构建体治疗癌症的疗效。拟议研究的更广泛影响旨在证明完全以碳水化合物为基础的疫苗如何在免疫系统中引起细胞和体液反应。该研究项目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。我们还期望这一方案广泛适用于疫苗研究的其他领域。

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.