Study of broadly neutralizing antibody generation to HIV gp140 in humanized mice

描述（由申请人提供）：获得具有广泛中和活性的抗HIV-1抗体的能力一直是HIV/AIDS疫苗研究的一个长期追求但难以实现的目标。该方案的长期目标是了解广谱中和抗体（BnAbs）是如何产生的。从hiv -1感染个体中分离的大多数罕见的BnAbs （2F5, 4E10和b12）具有长IGHV-CDR3s的共同特征，这些特征具有带正电的氨基酸，这是自身抗体中常见的结构特征。在这个提议中要测试的主要假设是，这种自身反应性抗体的自然诱导可能会被B细胞个体发生过程中发生的检查点控制机制所限制。因此，一个模拟人类免疫系统的系统，对HIV感染/免疫有反应，并且在B细胞检查点控制过程中具有可证明的缺陷，是一个有吸引力的平台来测试这一假设。通过3-辐照NOD/scid和NOD/scid IL2r？-/-小鼠，分别用胎儿肝脏和胸腺组织以及自体CD34+ hsc。人源化小鼠能够有效地植入多系人血淋巴样细胞，支持多产的HIV-1感染，在重组HIV gp140三聚体免疫后产生抗体反应，并表现出自身免疫特性。我们将测试这些小鼠在gp140 Env三聚体优化免疫后产生针对HIV-1的菌株特异性nAb和bnab的能力。为了实现这些目标，本提案的具体目的是：1)在HIVgp140免疫的BLT和GTL模型中，测试几种方法，包括添加人类细胞因子和/或活化T细胞，以诱导最佳抗原依赖类Ig转换和主要IgG反应。2)通过HIV假型病毒微量中和试验，探讨优化免疫是否产生中和抗体。3)通过克隆和表达FACS分选gp140-三聚体结合的b1样B细胞、常规B细胞（B2）和Ab分泌细胞(ASC)/浆细胞亚群的抗hiv包膜抗体（Env），来表征和定量表达中和抗体（nAb）应答的质量。从gp140-三聚体结合的B细胞克隆中获得的抗体的序列分析和结合/中和研究将提供详细的BnAbs分子特征。总的来说，我们的研究旨在开发一种新的小动物模型，以了解长期存在的BnAb如何产生的问题，并为产生此类BnAb应答的疫苗研究奠定基础。

DESCRIPTION (provided by applicant): The ability to elicit Abs with broad neutralization activity against HIV-1 has been a long sought-after but elusive goal of HIV/AIDS vaccine research. The long-term goal of this proposal is to understand how broad-spectrum neutralizing antibodies (BnAbs) are generated. The majority of the rare BnAbs (2F5, 4E10 and b12) isolated from HIV-1-infected individuals have the common characteristics of long IGHV-CDR3s with positively charged amino acids, structural features often seen in autoantibodies. The main hypothesis to be tested in this proposal is that natural induction of such autoreactive antibodies may be curtailed by the checkpoint control mechanisms occurring during B cell ontogeny. Therefore, a system that models a human immune system, is responsive to HIV infection/immunization and has demonstratable defects in B cell checkpoint control processes is an attractive platform to test this hypothesis. The humanized BLT and GTL mouse models are generated by transplantation of 3-irradiated NOD/scid and NOD/scid IL2r?-/- mice, respectively, with fetal liver and thymus tissues in addition to autologous CD34+ HSCs. The humanized mice allow efficient engraftment of multi-lineage human hemato-lymphoid cells, support a productive HIV-1 infection, develop Ab responses upon immunization with recombinant HIV gp140 trimer and demonstrate autoimmune properties. We will test the ability of these mice to generate strain-specific nAb and BnAbs against HIV-1 upon optimized immunization with gp140 Env trimers. Towards these goals, the specific aims of this proposal are: 1) To test several approaches including the addition of human cytokines and/or activated T cells to induce optimal antigen-dependent class Ig switching and a predominant IgG response in the HIVgp140 immunized BLT and GTL models. 2) To interrogate whether neutralizing antibodies are generated as a result of optimized immunization by HIV pseudotyped virus microneutralization assays. 3) To characterize and quantitate the quality of the neutralizing antibody (nAb) response by cloning and expressing anti-HIV Envelope (Env) Abs isolated by FACS sorting of gp140-trimer binding single B cells from B1-like, conventional B (B2) and Ab secreting cell (ASC)/plasma cell subpopulations. Sequence analysis and binding/neutralization studies of Abs derived from gp140-trimer binding B cell clones will provide a detailed molecular signature of BnAbs. Overall, our studies are aimed at developing a new small animal model to understand the long-standing question of how BnAbs are generated and to set the groundwork for vaccine studies that will result in generation of such BnAb responses. PUBLIC HEALTH RELEVANCE: A major reason for the failure of HIV vaccines is the lack of induction of broadly neutralizing anti-viral antibodies in the host. We propose to utilize new humanized mouse models to study the mechanism(s) by which BnAbs are produced and the barriers that must still be breached to elicit these types of antibodies by vaccination. The results of our studies will establish a new small animal HIV vaccine model and set the groundwork for vaccine studies aimed at the generation of potent and broadly neutralizing antibody responses to HIV.