Immunomodulation by exogenous streptococcal antibody

描述（由申请人提供）：在之前的资助周期中，我们鉴定了5种针对变形链球菌P1表面粘附素的单克隆抗体（mab），当与抗原作为免疫复合物一起施用时，这些单克隆抗体在数量、特异性和同型方面改变了小鼠的体液免疫反应。这一策略对疫苗设计具有启示意义，因为保护性免疫不一定针对病原体的免疫显性表位，并且可以通过将反应转向亚显性表位来很好地改善。结果取决于所检测的抗p1单抗。两种单克隆抗体在体外形成对变形链球菌粘附具有较强抑制作用的抗体，另外两种单克隆抗体形成的抑制作用较弱的抗体。一组抗p1单克隆抗体识别的表位具有特征，包括复杂的不连续决定因子。来自免疫小鼠的多克隆抗体也能识别构象表位，其中一些构象表位比其他构象表位更具生物学相关性。抗p1单克隆抗体抑制S. mutans粘附的能力与IgG2a和IgG2b亚型之间存在显著的正相关，这表明细胞因子在有益应答的发展过程中参与了类转换。这将作为当前提案的一部分加以探讨。在体外实验中，将抗P1单抗与细胞相关的P1结合可改变其对多种蛋白酶的易感性，这表明对蛋白质结构的影响，并提示暴露于隐表位以及抗原加工和呈递的变化。还将进行有关免疫调节这一潜在机制的研究。我们将继续描述免疫小鼠体液免疫反应中单克隆抗体介导的变化，以确定体外抗变形链球菌粘附和体内定植和致癌性的相关性。免疫小鼠样本的免疫分析将利用P1片段的组合或缺失结构，已知可以重建或破坏构象表位。迄今为止，尚未在接种疫苗的动物或自然致敏的人类中研究针对构象决定因素的抗p1反应。抗p1单克隆抗体的免疫调节代表了一种显著改善针对变形链球菌的体液反应的策略，并提供了一种工具来解剖针对这种广泛研究的候选疫苗抗原的有益和非有益抗体。抗P1单克隆抗体识别的表位的特征可以改善或减少有益的应答，这将为我们提供对诱导最佳保护性免疫所需的P1结构的深入了解，并有望揭示在不需要免疫复合物免疫的情况下修饰蛋白本身以提高其保护性免疫原性的方法。除了这些研究对人类龋齿治疗方法发展的影响之外，特性良好的P1抗原和针对它的单克隆抗体代表了一个很好的模型系统，可以理解免疫调节的后果和潜在的分子机制，并且对任何主动或被动免疫方法都有普遍的兴趣。

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.