Epitope shifting and Antibody Maturation during Rotavirus Infection

描述（由申请人提供）：我们将探索并确定表位移位的分子机制，这是最近被认识到的亲和成熟的特征，其中体细胞超突变改变了成熟抗体识别的表位，而不是种系抗体识别的表位。我们发表的研究表明，亲和成熟导致亲和和表位选择的变化，这两者对于产生中和抗体都是至关重要的。迄今为止，这一点尚未得到重视，因为先前的亲和力成熟研究主要集中在成熟提高对本质上相同的表位的亲和力的情况下。蛋白质抗原和半抗原都是如此，用蛋清溶菌酶和小半抗原进行的结构和生化研究就是例证。在这些情况下，亲和成熟改善了与相同表位的结合。抗溶菌酶抗体在这方面特别有趣，因为它们使用具有基本相同相互作用的大（~1,800E2）界面，其中亲和性增强源于相互作用表面边缘疏水表面的增加。这些变化是由非热点残留物的细微重排驱动的。而抗溶菌酶抗体的相互作用表面的边缘发生变化，这些变化不会导致识别的表位发生变化。在我们的模型中，这是因为热点残基是种系编码的。我们提出的工作将探索假设发生在抗体结合位点的变化，并建立这些变化导致表位移动的机制。我们将重点关注使用VH1-46基因片段的抗轮状病毒VP6抗体。VP6是轮状病毒抗体应答的免疫显性表位，VH1-46是轮状病毒抗体应答的显性重链基因片段。一般来说，成年人会产生轮状病毒的中和抗体，但婴儿不会。这不是由于种系基因的使用，而是由于成人中更多的体细胞超突变。轮状病毒是发达国家和发展中国家婴幼儿严重腹泻的最重要原因。已经引入了减毒病毒疫苗，然而，即使在控制环境中适当接种，这些疫苗似乎也只能减少约61%的感染。此外，它们不能对新出现的菌株提供保护，并且需要多次（2或3）剂量。人们一直在寻找疗效更好的第二代疫苗，亚单位疫苗是一个很有吸引力的选择。疾控中心估计，即使接种了疫苗，每年仍有50万人死于轮状病毒感染。亚单位疫苗的有效开发得益于对人体免疫系统识别抗原和中和病原体的机制的全面了解。这一应用将提供可能影响抗原选择、替代保护标记的使用和新型轮状病毒疫苗开发的分子免疫学范式。我们将探索和确定表位移位的分子基础。

DESCRIPTION (provided by applicant): We will explore and determine the molecular mechanism for epitope shift, a recently recognized feature of affinity maturation in which somatic hypermutation changes the epitope recognized by mature antibodies from that recognized by germline antibodies. Our published work indicates that affinity maturation leads to both affinity and epitope selection changes, both of which can be critical for developing neutralizing antibodies. To date, this has been unappreciated because previous studies of affinity maturation have focused on cases where maturation improves affinity to an essentially identical epitope. This is the case both for protein antigens and haptens, as exemplified by structural and biochemical studies using hen egg white lysozyme and small haptens. In these cases, affinity maturation improves binding to identical epitopes. The anti-lysozyme antibodies are particularly interesting in this light, because they use large (~1,800E2) interfaces with essentially identical interactions where enhanced affinity arises from increased burial of hydrophobic surface at the edge of the interaction surface. These changes are driven by subtle rearrangements of non-hotspot residues. While the edge of the interaction surface changes in the anti- lysozyme antibodies, these changes do not lead to changes in the epitope recognized. In our model, this is because hot-spot residues are germline encoded. Our proposed work will explore changes hypothesized to occur within the antibody combining site and establish the mechanism whereby these changes cause epitope shifting. We will focus anti-rotavirus VP6 antibodies that use the VH1-46 gene segment. VP6 is the immunodominant epitope and VH1-46 is the dominant heavy chain gene segment seen in the antibody response against rotavirus. In general, adults produce neutralizing antibodies to rotavirus, but infants do not. This is not due to germline gene usage, but rather it is to more somatic hypermutation in adults. Rotaviruses are the most important cause of severe diarrhea in infants and young children in both the developed world and the developing world. Attenuated virus vaccines have been introduced, however even when properly administered in a controlled setting, these vaccines appear to reduce infection by only ~61%. Additionally, they offer no protection against emerging strains, and require multiple (2 or 3) doses. Second generation vaccines with improved efficacy are sought, and subunit vaccines are an attractive choice. CDC estimates are that even with vaccination ~500,000 people still die annually from rotavirus infection. Effective development of subunit vaccines is aided by a comprehensive understanding of the mechanisms the human immune system employs to recognize antigens and neutralize pathogens. This application will provide molecular immunology paradigms that may influence the choice of antigens, the use of surrogate markers of protection, and the development of new rotavirus vaccines. We will explore and determine the molecular basis for epitope shift. PUBLIC HEALTH RELEVANCE: Despite recent vaccines, rotaviruses remain the largest cause of severe diarrhea, with estimates indicating that vaccines are likely 45-61% effective, the CDC estimates that rotavirus still claims >500,000 lives annually, mostly in children younger than 5 years in the developing world. The antibody immune response to this disease is impaired in infants, such that infants use the same dominant germline genes, but have impaired affinity maturation processes and do not produce neutralizing antibodies. We will determine the mechanism of a newly identified aspect of affinity maturation, epitope shift, and provide new molecular immunology paradigms that may influence the choice of antigens, the use of surrogate markers of protection, and the development of new rotavirus vaccines.