GENETICALLY ENGINEERED ORAL VACCINES AND CARIES IMMUNITY

龋齿是一种主要病原体 变形链球菌和远缘链球菌。研究 旨在诱导对传染病的免疫力，包括牙科疾病， 龋，提供了宝贵的信息，微生物抗原的重要 在诱导保护性反应;在防御伊加抗体的作用 防止感染侵入或定植于外部环境的表面 分泌物;以及诱导免疫反应的机制。 与这些研究同时，重组DNA的进展 技术和基因克隆导致了更多关于 微生物的基因产物在毒力和发展的重要性， 新型疫苗本项目的总体目标是确定机制 由重组无毒沙门氏菌组成的粘膜疫苗， 表达变异链球菌克隆基因的菌株，有和没有 佐剂，诱导特异性免疫应答。具体而言，我们将：l） 确定沙门氏菌表达的克隆B亚单位的有效性 霍乱毒素（CTB）在促进共表达克隆 变形链球菌抗原。抗体水平和同种型 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测量，以确定免疫原性和佐剂性质， 抗原与CTB在体内的不同融合类型。分析 在大鼠模型中的龋齿活动将评估 应答这些研究将确定哪种结构类型的嵌合 当沙门氏菌在体内表达蛋白质时， 促进唾液伊加抗体应答，保护性对抗变异体 链球菌。2)确定沙门氏菌疫苗株的效果， 载体和启动子对变异株克隆抗原表达的影响 链球菌和诱导免疫应答。的量 沙门氏菌突变株产生的变异链球菌克隆抗原 含有具有不同启动子的表达载体的菌株， 通过ELISA测量复制子。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免疫。水平和同种型 血清和外分泌物中克隆抗原的抗体 用沙门氏菌菌株口服免疫的动物，所述沙门氏菌菌株含有不同的 将测量矢量，以确定是否存在差异， 粘膜免疫应答的强度和持续时间以及保护作用 抵抗变异链球菌的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 通过克隆的蛋白质的体内表达诱导， 组成型启动子（trc）与在厌氧环境中诱导的启动子 环境（nirB）具有更大的幅度和更长的持续时间。第三章 确定重组免疫途径的效果 沙门氏菌疫苗的规模和保护能力， 唾液伊加抗体应答。伊加抗体水平的克隆 通过口服或鼻内途径免疫的动物唾液中的抗原 将通过ELISA测量以确定哪种免疫途径诱导 最高的唾液伊加抗体反应保护免受感染， 变形链球菌这些研究的结果将与以下方面有关： 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应答 包括与口腔相关的那些。

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.