Modeling affinity maturation at molecular resolution

 描述（由申请方提供）：诱导有效的亲和力成熟是有效体液疫苗的重要组成部分。最近在流感和HIV-1疫苗开发方面的发现表明，在这些关键领域的成功将取决于影响亲和力成熟的能力，这种影响达到目前尚不可能的程度。在实验方法的不断进步，阐明细胞的相互作用，在germinal中心的反应，现在有可能理解和模型选择的亲和力成熟。具体目标围绕着通过实验、开发、预测和验证周期开发和使用数学模型的主要目标进行组织。对于所有研究目的，使用的抗体和抗体克隆将从人和鼠疫苗研究中分离和表征的抗体和抗体克隆中选择。所开发的模型将包括体细胞超突变序列特异性突变率的精确模型。通过使用经工程改造以生产性和非生产性但突变形式表达相同重排的IG基因的小鼠，该模型将成为可能。该模型将包含氨基酸突变之间的对应关系的统计学准确模型和由于这些突变引起的亲和力变化。这些模型将通过使用新的高通量B细胞培养系统来实现，该系统允许从单个B细胞中分离轻链/重链对以及测量由这些基因编码的抗体的亲合力。此外，我们将获得所选克隆相关抗体的结构和详细的动力学结合参数。这些研究中使用的IG可变区基因将选自在人类对季节性流感疫苗应答的亲和力成熟研究和流感病毒血凝素或炭疽杆菌保护性抗原免疫的小鼠研究中分离的基因。然后，这些研究中获得的模型和方法将用于研究1）在使用对照小鼠研究的重复实验中以及在人类受试者中的重复接种中亲和力成熟的重要特征被保留的程度;和2）调节有效选择强度对亲和力成熟的影响。 过表达抗凋亡基因的小鼠中。我们将在我们自己的实验室，并通过暑期学校和研讨会提供跨学科的培训。所有模型和软件将与科学界共享。

 DESCRIPTION (provided by applicant): Eliciting effective affinity maturation is an essential component of efficacious humoral vaccines. Recent findings in vaccine development for influenza and HIV-1 suggest that success in these key areas will depend on the ability to influence affinity maturation to an extent not yet possible. Continuing advances in experimental methods for elucidating cellular interactions in the germinal center reaction now make it possible to understand and model selection in affinity maturation. The specific aims are organized around the major goal of developing and using a mathematical model through cycles of experimentation, development, prediction and validation. For all research aims, the antibodies and antibody clones used will be selected from among those isolated and characterized in human and murine vaccine studies. The model developed will include an accurate model of somatic hypermutation sequence- specific mutation rate. This model will be made possible through the use of mice engineered to express the same rearranged Ig gene in productive and non-productive, but mutating, forms. The model will contain statistically accurate models of the correspondence between amino acid mutations and changes in affinity due to these mutations. These models will be realized through the use of a novel high-throughput B-cell culture system that allows the isolation of light- chain/heavy-chain pairs from individual B cells as well as measurement of the avidity of the antibody encoded by these genes. Furthermore, we will obtain the structures and detailed kinetic binding parameters for selected clonally related antibodies. The Ig variable-region genes to be used in these studies will be selected from genes isolated in a study of affinity maturation in the human response to seasonal influenza vaccine and in murine studies of influenza virus hemagglutinin or Bacillus anthracis protective antigen immunization. The models and methods obtained in these studies will then be used to study 1) the extent to which important features of affinity maturation are conserved in repeated experiments using controlled mouse studies as well as repeated vaccinations in human subjects; and 2) the effects on affinity maturation of modulating the effective selection intensity in mice engineered to overexpress an anti-apoptotic gene. We will provide cross-disciplinary training in our own laboratories and through summer schools and symposia. All models and software will be shared with the scientific community.