GENETICALLY ENGINEERED ORAL VACCINES AND CARIES IMMUNITY

基因工程口服疫苗和龋齿免疫

• 批准号: 2872148
• 负责人: Suzanne M. Michalek
• 金额: $ 23.3万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-02-01 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2872148
• 关键词: Peyer's patches; Salmonella; Salmonella vaccines; Streptococcus mutans; bacterial antigens; biotechnology; cholera toxin; dental caries vaccine; drug administration rate /duration; electron microscopy; enzyme linked immunosorbent assay; genetic promoter element; gram negative bacteria; immunofluorescence technique; immunogenetics; immunomodulators; laboratory rat; lymphocyte; molecular cloning; mucosal immunity; nonhuman therapy evaluation; oral administration; recombinant DNA; transfection /expression vector; vaccine development; vector vaccine

Dental caries is an infectious disease in which the major etiologic agents in humans are Streptococcus mutans and Streptococcus sobrinus. Studies aimed at inducing immunity against infectious diseases, including dental caries, have provided valuable information on microbial antigens important in inducing protective responses; on the role of IgA antibodies in defense against infections which invade or colonize surfaces bathed by external secretions; and on the mechanisms involved in inducing immune responses. Concurrent with these studies, the advancements in recombinant DNA technology and gene cloning have led to additional information on microbial gene products important in virulence and to the development of novel vaccines. The overall goal of this project is to define mechanisms by which mucosal vaccines consisting of recombinant, avirulent Salmonella strains expressing cloned genes of mutans streptococci, with and without adjuvant, induce specific immune responses. Specifically, we will: l) Determine the effectiveness of Salmonella expressed cloned B subunit of cholera toxin (CTB) in promoting immune responses to co-expressed cloned antigens of mutans streptococci. The levels and isotype of antibodies to the cloned antigens in serum and external secretions of mice and rats immunized orally with Salmonella expressing chimeric proteins will be measured by ELISA to determine the immunogenic and adjuvant properties of the different types of fusions between antigen and CTB in vivo. Analysis of caries activity in a rat model will assess the protective effect of the responses. These studies will determine which structural type of chimeric protein, when expressed by Salmonella in vivo, is most effective in promoting salivary IgA antibody responses protective against mutans streptococci. 2) Determine the effect of the Salmonella vaccine strain, vector, and promoter on the expression of cloned antigen of mutans streptococci and on the induction of immune responses. The amount of cloned antigen of mutans streptococci produced by Salmonella mutant strains containing expression vectors with different promoters and replicons will be measured by ELISA. Stability of the expression vectors will be assessed by microbiologic detection of Salmonella in tissues of mice orally-immunized with the Salmonella vaccine. The level and isotype of antibodies to the cloned antigen in serum and external secretions of animals orally immunized with a Salmonella strain containing different vectors will be measured to determine if differences exist in the magnitude and duration of the mucosal immune responses and in protection against infection by mutans streptococci. These studies will determine the effect of the vector replicon on the in vivo expression of cloned protein and on the induction of immune responses, and whether immune responses induced by the in vivo expression of cloned protein under the control of a constitutive promoter (trc) versus one induced in an anaerobic environment (nirB) are of greater magnitude and longer duration. 3) Determine the effect of route of immunization with a recombinant Salmonella vaccine on the magnitude and protective capability of the salivary IgA antibody response. The level of IgA antibodies to the cloned antigen in saliva in animals immunized by the oral or intranasal route will be measured by ELISA to determine which route of immunization induces the highest salivary IgA antibody response protective against infection by mutans streptococci. The results of these studies will be relevant in establishing the practicability of Salmonella vaccine delivery systems for the induction of protective immune responses against mucosal pathogens including those associated with the oral cavity.

龋齿是一种传染性疾病，其主要病因是 人类中的链球菌是变形链球菌和远缘链球菌。研究 旨在诱导对传染病（包括牙科疾病）的免疫力 龋齿，提供了有关微生物抗原的重要信息 诱导保护性反应； IgA 抗体在防御中的作用 防止侵入或定植于外部沐浴表面的感染 分泌物；以及诱导免疫反应的机制。 与这些研究同时进行的还有重组 DNA 的进展 技术和基因克隆带来了更多关于 微生物基因产物对毒力和发展具有重要意义 新型疫苗。该项目的总体目标是定义机制 由重组、无毒力沙门氏菌组成的粘膜疫苗 表达变形链球菌克隆基因的菌株，有或没有 佐剂，诱导特异性免疫反应。具体来说，我们将： l) 确定沙门氏菌表达的克隆 B 亚基的有效性 霍乱毒素（CTB）促进共表达克隆的免疫反应 变形链球菌抗原。抗体的水平和同种型 小鼠和大鼠血清和外分泌物中的克隆抗原 用表达嵌合蛋白的沙门氏菌口服免疫 通过 ELISA 测定以确定免疫原性和佐剂特性 体内抗原和CTB之间不同类型的融合。分析 大鼠模型中的龋齿活动将评估该材料的保护作用 回应。这些研究将确定嵌合体的结构类型 当沙门氏菌在体内表达蛋白质时，最有效 促进唾液 IgA 抗体反应，预防变异病毒 链球菌。 2) 确定沙门氏菌疫苗株的效果， 表达突变体克隆抗原的载体和启动子 链球菌和诱导免疫反应。金额 沙门氏菌突变体产生的变形链球菌克隆抗原 含有具有不同启动子的表达载体的菌株和 复制子将通过 ELISA 进行测量。表达载体的稳定性 将通过组织中沙门氏菌的微生物检测进行评估 小鼠口服沙门氏菌疫苗进行免疫。水平和同种型 血清和外分泌物中克隆抗原的抗体 用含有不同成分的沙门氏菌菌株口服免疫的动物 将测量向量以确定是否存在差异 粘膜免疫反应的强度和持续时间以及保护作用 抵抗变形链球菌感染。这些研究将决定 载体复制子对克隆蛋白体内表达的影响 以及免疫反应的诱导，以及免疫反应是否 由克隆蛋白在体内表达的控制下诱导 组成型启动子 (trc) 与厌氧条件下诱导的启动子 环境（nirB）的强度更大，持续时间更长。 3） 确定重组免疫途径的效果 沙门氏菌疫苗对沙门氏菌感染程度和保护能力的影响 唾液 IgA 抗体反应。克隆的 IgA 抗体水平 通过口服或鼻内途径免疫的动物唾液中的抗原 将通过 ELISA 进行测量以确定哪种免疫途径诱导 最高的唾液 IgA 抗体反应可防止感染 变形链球菌。这些研究的结果将与 建立沙门氏菌疫苗输送系统的实用性 诱导针对粘膜病原体的保护性免疫反应 包括与口腔有关的那些。