Immunomodulation by exogenous streptococcal antibody

描述（由申请人提供）： 在上一个资助周期中，我们确定了五种针对变形链球菌P1表面粘附素的单克隆抗体（MAbs），当与抗原作为免疫复合物一起施用时，这些抗体在引发抗体的数量、特异性和同种型方面重定向小鼠中的体液免疫应答。这一策略对疫苗设计有影响，因为保护性免疫不一定针对病原体的免疫显性表位，并且可以通过将应答转向亚显性表位来改善。结果因检测的抗P1单克隆抗体而异。两种单克隆抗体均能产生对沙门氏菌有较强抑制作用的抗体。另外两种是抑制性较小的抗体。由一组抗P1单克隆抗体识别的表位进行了表征，包括复杂的不连续决定簇。来自免疫小鼠的多克隆抗体也识别构象表位，其中一些表位似乎比其他表位更具有生物学相关性。抗P1单克隆抗体抑制S.变形杆菌粘附和IgG2a和IgG2b同种型表明参与类转换的细胞因子在有益应答的发展中的相关性。这将作为本提案的一部分加以探讨。抗P1单克隆抗体与细胞相关P1的结合改变了其对体外多种蛋白酶的敏感性，表明对蛋白质结构的影响，并表明隐藏表位的暴露以及抗原加工和呈递的变化。还将进行研究，以解决这种潜在的免疫调节机制。我们将继续研究单克隆抗体介导的免疫小鼠体液免疫应答的变化，以确定抗沙门氏菌保护的相关性。体外的变形菌粘附和体内的定植和致龋性。来自免疫小鼠的样品的免疫测定将利用已知重建或破坏构象表位的P1区段或缺失构建体的组合。迄今为止，尚未在接种疫苗的动物或自然致敏的人中研究针对构象决定簇的抗P1应答。抗P1单克隆抗体的免疫调节是一种显著提高抗沙门氏菌体液免疫应答的策略。变异的，并提供了一种工具，剖析有益的和非有益的抗体，对这种广泛研究的候选疫苗抗原。抗P1单克隆抗体识别的表位的表征，提高或降低有益的反应将提供深入了解P1的结构，需要引发最佳的保护性免疫，并有望阐明如何修改蛋白质本身，以提高其保护性免疫原性，而不需要免疫复合物。除了这些研究对开发人类龋齿治疗方法的影响外，充分表征的P1抗原和抗P1抗原的单克隆抗体代表了理解免疫调节的后果和潜在分子机制的极好模型系统，并且对于任何主动或被动免疫方法都具有普遍意义。

DESCRIPTION (provided by applicant): During the previous funding cycle we identified five monoclonal antibodies (MAbs) against the P1 surface adhesin of Streptococcus mutans that redirect the humoral immune response in mice in terms of quantity, specificity and isotype of elicited antibodies when administered with the antigen as an immune complex. This strategy has implications for vaccine design in that protective immunity is not necessarily directed at immunodomiant epitopes of pathogens and could well be improved by shifting a response toward subdominant epitopes. Results varied depending on the anti-P1 MAb tested. Two MAbs resulted in formation of antibodies more inhibitory of S. mutans adherence in vitro and two others of less inhibitory antibodies. Epitopes recognized by a panel of anti-P1 MAbs were characterized and include complex discontinuous determinants. Polyclonal antibodies from immunized mice also recognize conformational epitopes, some of which appear more biologically relevant than others. Significant positive correlations were demonstrated between the ability of anti-P1 MAbs to inhibit S. mutans adherence and IgG2a and IgG2b isotypes suggesting the relevance of cytokines involved in class switching in the development of beneficial responses. This will be explored as part of the current proposal. Binding of an anti-P1 MAb to cell-associated P1 altered its susceptibility to numerous proteases in vitro indicating an influence on protein structure and suggesting exposure of cryptic epitopes and changes in antigen processing and presentation. Studies to address this potential mechanism of immunomodulation will also be undertaken. We will continue to characterize MAb-mediated changes in humoral immune responses in immunized mice to identify correlates of protection against S. mutans adherence in vitro and colonization and cariogenicity in vivo. Immunoassays of samples from immunized mice will utilize combinations of P1 segments, or deletion constructs, known to reconstitute or destroy conformational epitopes. The anti-P1 response against conformational determinants has not been studied to date in vaccinated animals or naturally sensitized humans. Immunomodulation by anti-P1 MAbs represents a strategy to significantly improve the humoral response against S. mutans and provides a tool to dissect beneficial and non-beneficial antibodies against this widely studied candidate vaccine antigen. The characterization of epitopes recognized by anti-P1 MAbs that improve or decrease the beneficial response will provide insight into the structure of P1 required to elicit optimal protective immunity and is expected to shed light on ways to modify the protein itself to improve its protective immunogenicity without the need to immunize with immune complexes. Beyond the impact of these studies on development of a therapeutic approach against human dental caries, the well-characterized P1 antigen and MAbs against it represent an excellent model system to understand the consequences and underlying molecular mechanisms of immunomodulation and would be of general interest for any active or passive immunization approach.