Role of IG CDR-H3 in Responses to HIV Vaccines

描述（由申请人提供）： 在针对HIV-1包膜gp 120/gp 41糖蛋白的中和抗体（Nab）中，2F 5和4 E10因其效力和广泛中和活性而脱颖而出。两种抗体均针对gp 41茎的膜近端外部区域（MPER）。不幸的是，2F 5和4 E10样抗MPER Nab很少是抗HIV应答的主要成分。2F 5和4 E10与标准的不同之处在于，它们的抗原结合位点中的每一个含有长的H链互补决定区3（CDR-H3），其缺乏酪氨酸并且包括疏水性和带电氨基酸的补丁。在先前的研究中，我们已经表明，携带带有带电或疏水性CDR-H3的免疫球蛋白的B细胞通常从滤泡和成熟的再循环B细胞亚群中剔除，即最可能从中提取T依赖性抗体应答的亚群。我们建议测试2F 5和4 E10样Nab是否难以在健康个体中引发，因为通过遗传和体细胞机制，表达具有这种类型抗原结合位点的Ig的B细胞的频率被故意保持在较低水平。我们将通过使用基因靶向以用编码2F 5或4 E10的CDR-H3序列的DH替换DH基因座来实现这一点。这将迫使表达2F 5和4 E10样CDR-H3的抗体谱的表达。然后，我们将测试使用2F 5和4 E10衍生的CDR-H3间隔的强制富集是否会促进针对HIV-1 MPER的Nab的产生。为了检验互补假设，即在抗MPER Nab中使用2F 5-和4 E10-衍生的CDR-H3间隔可能需要从库控制的正常机制中释放，我们将繁殖这些小鼠突变，这些突变改变B细胞信号传导、库选择或细胞命运结果的模式。该申请是响应RFA的，因为它专门设计用于确定“为什么感染引起的广泛中和抗体是罕见的，以及免疫原如何引发它们”，并开发“更相关的动物模型，其中可以探索保护或增强感染。我们的研究还涉及“调节和/或特异性抑制免疫反应的组成部分以改变疾病进展”，以确定如何操纵免疫反应以最佳地产生针对HIV的保护性抗体。这些研究还将促进UAB的三位基础B细胞免疫学家Schroeder博士、Justement博士和Kearney博士以及杜克大学的Haynes博士、加州大学洛杉矶分校的Grovit-Ferbas博士和UAB的Shaw博士这三位艾滋病专家之间的合作。事实证明，制造能够引发中和抗体的疫苗比最初预期的要困难得多，因为许多广泛中和的抗体不仅极其罕见，而且往往具有自我反应性。我们建议在小鼠中使用基因靶向来迫使B细胞，包括滤泡亚群，优先表达富含这种类型抗体的抗体库，然后测试是否改变BCR信号传导，库选择或免疫原类型（例如脂质体包裹的抗原或假病毒体展示的抗原）将促进抗体的产生，这些抗体可以结合HIV包膜蛋白的关键MPER区域，从而使它们能够广泛地中和HIV。

DESCRIPTION (provided by applicant): Among neutralizing antibodies (Nabs) directed against the HIV-1 envelope gp120/gp41 glycoprotein, 2F5 and 4E10 stand out for their potency and broadly neutralizing activity. Both antibodies are directed against the membrane proximal external region (MPER) of the gp41 stalk. Unfortunately, 2F5 and 4E10-like anti-MPER Nabs are rarely major components of the anti-HIV response. 2F5 and 4E10 differ from the norm in that each of their antigen binding sites contain a long, H chain complementarity determining region 3 (CDR-H3) that lacks tyrosine and includes patches of hydrophobic and charged amino acids. In previous studies we have shown that B cells bearing immunoglobulins with charged or hydrophobic CDR-H3s are normally culled from the follicular and mature, recirculating B cell subsets, i.e. the subsets from which T-dependent antibody responses are most likely to be drawn. We propose to test whether 2F5- and 4E10-like Nabs are difficult to elicit in healthy individuals because the frequency of B cells expressing Igs with this type of antigen binding site is deliberately kept low by both genetic and somatic mechanisms. We will do this by using gene targeting to replace the DH locus with a DH encoding the CDR-H3 sequence of either 2F5 or 4E10. This will force expression of a spectrum of antibodies expressing 2F5 and 4E10-like CDR-H3s. We will then test whether forced enrichment for the use of 2F5- and 4E10-derived CDR-H3 intervals will promote the production of Nabs directed against the HIV-1 MPER. To test the complementary hypothesis that the use of 2F5- and 4E10-derived CDR-H3 intervals in anti-MPER Nabs may require release from normal mechanisms of repertoire control, we will breed into these mice mutations that alter patterns of B cell signaling, repertoire selection, or cell fate outcomes. The application is responsive to the RFA in that it is specifically designed to determine "why broadly neutralizing antibodies that arise from infection are rare and how immunogens can elicit them" and to develop "more relevant animal models in which protection or enhancement of infection can be explored." Our studies also touch on "Modulating and/or specifically suppressing component aspects of the immune response to alter disease progression" in the context of determining how to manipulate the immune response in order to optimally generate protective antibodies against HIV. These studies will also serve to foster collaboration between three established basic B cell immunologists at UAB, Drs. Schroeder, Justement and Kearney; and three experts in HIV, Drs. Haynes at Duke, Grovit-Ferbas at UCLA, and Shaw at UAB. The creation of vaccines capable of eliciting neutralizing antibodies to HIV has proven more difficult than first expected because many of the antibodies that are broadly neutralizing are not only extremely uncommon; they also tend to be self-reactive. We propose to use gene targeting in mice to force B cells, including the follicular subset, to preferentially express antibody repertoires enriched for antibodies of this type, and then test whether altering BCR signaling, repertoire selection or the type of immunogen (e.g. antigen encased in liposomes or antigen displayed by pseudovirions) will facilitate the generation of antibodies that can bind the key MPER region of the HIV envelope protein in a way that will enable them to broadly neutralize HIV.