Modeling affinity maturation at molecular resolution

 描述(由申请人提供)：诱导有效的亲和力成熟是有效的体液疫苗的重要组成部分。流感和HIV-1疫苗开发的最新发现表明，这些关键领域的成功将取决于影响亲和力成熟的能力，而这种影响的程度尚不可能。在生发中心反应中阐明细胞相互作用的实验方法的不断进步现在使得理解亲和力成熟和模型选择成为可能。具体目标是围绕通过实验、开发、预测和验证的循环开发和使用数学模型这一主要目标来组织的。出于所有研究目的，所使用的抗体和抗体克隆将从人类和小鼠疫苗研究中分离和表征的抗体和抗体克隆中挑选出来。开发的模型将包括一个精确的体细胞超突变序列特异性突变率模型。这个模型将通过使用经过改造的小鼠来实现，这些小鼠能够以生产性和非生产性的形式表达相同的重排免疫球蛋白基因，但会发生突变。该模型将包含氨基酸突变与这些突变引起的亲和力变化之间的对应关系的统计准确模型。这些模型将通过使用一种新的高通量B细胞培养系统来实现，该系统允许从单个B细胞中分离出轻链/重链对，并测量这些基因编码的抗体的亲和力。此外，我们将获得选定的克隆性相关抗体的结构和详细的动力学结合参数。这些研究中使用的免疫球蛋白可变区基因将从人类对季节性流感疫苗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出来。在这些研究中获得的模型和方法将被用来研究1)在使用受控小鼠研究和在人类受试者中重复接种疫苗的重复实验中，亲和力成熟的重要特征被保留的程度；以及2)调节有效选择强度对亲和力成熟的影响 在基因改造的小鼠中过度表达一种抗凋亡基因。我们将在我们自己的实验室以及通过暑期学校和研讨会提供跨学科培训。所有模型和软件都将与科学界共享。

 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.