Immunomodulation by exogenous streptococcal antibody

描述(由申请人提供)：在前一个资助周期中，我们鉴定了五种针对变形链球菌P1表面粘附素的单抗(MAb)，当与该抗原作为免疫复合体注射时，这些单抗在数量、特异性和同种类型方面重定向了小鼠的体液免疫反应。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优势表位，而可以通过将反应转向次要表位来改善。结果因抗P1单抗的不同而不同。其中两株单抗对变形链球菌的体外黏附有较强的抑制作用，另两株对变形链球菌的体外黏附抑制作用较弱。对一组抗P1单抗识别的表位进行了表征，其中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，其中一些似乎比另一些更具生物学相关性。抗P1单抗抑制变形链球菌黏附的能力与IgG2a和IgG2b亚型呈显着正相关，提示参与类别转换的细胞因子在有益反应的发展中具有相关性。这将作为当前提案的一部分进行探讨。抗P1单抗与细胞相关的P1结合，在体外改变了其对多种蛋白酶的敏感性，表明对蛋白质结构的影响，并提示隐蔽表位的暴露和抗原处理和提呈的改变。还将进行研究以解决这一潜在的免疫调节机制。我们将继续研究单抗介导的免疫小鼠体液免疫反应的变化，以确定体外对变形链球菌黏附的保护与体内定植和致龋性的相关性。免疫小鼠样本的免疫分析将利用已知的重建或破坏构象表位的P1片段或缺失结构的组合。到目前为止，在接种疫苗的动物或自然致敏的人类中还没有研究过针对构象决定因素的抗P1反应。抗P1单抗的免疫调节代表了一种显著提高对变形链球菌的体液反应的策略，并提供了一种工具来分析针对这种被广泛研究的候选疫苗抗原的有益和无益抗体。对抗P1单抗识别的可改善或减少有益反应的表位的表征将有助于深入了解诱导最佳保护性免疫所需的P1的结构，并有望阐明如何修饰蛋白质本身以提高其保护性免疫原性，而不需要免疫复合体免疫。除了这些研究对开发一种治疗人类龋齿的方法的影响外，具有良好特性的P1抗原和针对它的单抗是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.