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转换的分子机制，表位转换是最近认识到的亲和力成熟特征，其中体细胞超突变将成熟抗体识别的表位从种系抗体识别的表位改变。我们发表的工作表明，亲和力成熟导致亲和力和表位选择的变化，这两者对于开发中和抗体都是至关重要的。迄今为止，这还没有得到重视，因为先前的亲和力成熟研究集中在成熟提高对基本相同表位的亲和力的情况下。这是蛋白质抗原和半抗原的情况，如使用鸡蛋白色溶菌酶和小半抗原的结构和生物化学研究所例示的。在这些情况下，亲和力成熟改善与相同表位的结合。抗溶菌酶抗体在这方面特别令人感兴趣，因为它们使用具有基本相同的相互作用的大（~1，800 E2）界面，其中增强的亲和力来自于在相互作用表面的边缘处疏水表面的增加的掩埋。这些变化是由非热点残基的细微重排驱动的。虽然在抗溶菌酶抗体中相互作用表面的边缘发生变化，但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讨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转移的机制。我们将重点关注使用VH 1 -46基因片段的抗轮状病毒VP 6抗体。 VP 6是免疫显性表位，VH 1 -46是在针对轮状病毒的抗体应答中看到的显性重链基因片段。一般来说，成人会产生轮状病毒的中和抗体，但婴儿不会。这不是由于生殖系基因的使用，而是由于成年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，但是即使在受控环境中适当施用，这些疫苗似乎也只能减少约61%的感染。此外，它们对新出现的菌株没有保护作用，需要多次（2或3次）给药。人们正在寻求具有改善的效力的第二代疫苗，亚单位疫苗是一种有吸引力的选择。CDC估计，即使接种疫苗，每年仍有约50万人死于轮状病毒感染。 对人类免疫系统识别抗原和中和病原体的机制的全面理解有助于亚单位疫苗的有效开发。该应用将提供分子免疫学范例，其可能影响抗原的选择、保护的替代标志物的使用以及新轮状病毒疫苗的开发。我们将探索和确定表位转移的分子基础。 公共卫生相关性：尽管最近的疫苗，轮状病毒仍然是严重腹泻的最大原因，估计表明疫苗可能有45-61%的有效性，CDC估计轮状病毒每年仍然夺去> 500，000人的生命，主要是发展中国家5岁以下的儿童。婴儿对这种疾病的抗体免疫应答受损，因此婴儿使用相同的优势种系基因，但亲和力成熟过程受损，不产生中和抗体。我们将确定一个新确定的亲和力成熟，表位转移方面的机制，并提供新的分子免疫学范式，可能会影响抗原的选择，使用替代标志物的保护，和新的轮状病毒疫苗的开发。

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.