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gp120/gp41糖蛋白的中和抗体中，2F5和4E10以其效力和广泛的中和活性而脱颖而出。这两种抗体都是针对gp41茎的膜近端外部区域(MPER)的。不幸的是，2F5和4E10类抗MPER NAB很少是抗HIV反应的主要成分。2F5和4E10的不同之处在于，它们的每个抗原结合部位都含有一个长的H链互补决定区3(CDR-H3)，该区域缺乏酪氨酸，包括片状的疏水和带电氨基酸。在以前的研究中，我们已经证明，携带带有带电或疏水CDR-H3的免疫球蛋白的B细胞通常从滤泡和成熟的循环B细胞亚群中剔除，即最有可能产生T依赖抗体反应的亚群。我们建议测试2F5和4E10样NAB是否很难在健康个体中诱导，因为具有这种抗原结合位点的B细胞表达IGs的频率被遗传和躯体机制故意保持在低水平。为此，我们将使用基因打靶技术，用编码2F5或4E10的CDR-H3序列的水解酶来取代水解酶基因。这将强制表达一系列表达2F5和4E10样CDR-H3的抗体。然后，我们将测试强制浓缩使用2F5和4E10衍生的CDR-H3间隔是否会促进针对HIV-1 MPER的NAB的产生。为了检验互补假设，即在抗MPER NAb中使用2F5和4E10衍生的CDR-H3间隔可能需要从正常的谱系控制机制中释放，我们将在这些小鼠中培育改变B细胞信号模式、谱系选择或细胞命运结果的突变。这项应用是对RFA的响应，因为它是专门设计来确定“为什么感染产生的广泛中和抗体很少见，以及免疫原如何诱导它们”，并开发“可以探索感染保护或增强的更相关的动物模型。”我们的研究还涉及在确定如何操纵免疫反应以便以最佳方式产生针对艾滋病毒的保护性抗体的背景下，“调节和/或具体抑制免疫反应的组成部分，以改变疾病的进展”。这些研究还将促进UAB的三位知名基础B细胞免疫学家Schroeder、Just ement和Kearney博士与杜克大学的Haynes博士、加州大学洛杉矶分校的Grovit-Ferbas博士和UAB的Shaw博士之间的合作。事实证明，能够产生艾滋病毒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能力。我们建议在小鼠中使用基因打靶来迫使B细胞，包括滤泡亚群，优先表达针对这类抗体的抗体谱系，然后测试改变BCR信号、谱系选择或免疫原类型(例如脂质体中的抗原或伪病毒显示的抗原)是否会促进抗体的产生，这些抗体可以结合HIV包膜蛋白的关键MPER区域，使它们能够广泛中和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.