MPER broadly neutralizing antibody knockin mice to study anti-HIV Bcell responses

描述(由申请人提供)：几种罕见的针对不同HIV-1包膜表位的抗体的鉴定，能够在体外中和广泛的HIV-1分离株，并在体内控制SIV感染，为实现基于抗体的HIV-1疫苗策略提供了希望。其中一种罕见的抗体，2F5，具有特别有效和广泛的中和活性，并针对基于抗体的疫苗靶标，HIV-1 gp41区域的MPER表位。2F5具有异常长的带电CDR3，并表现出多反应性，这增加了像2F5这样的抗体无法激发的可能性，因为它们起源的B细胞与宿主抗原具有反应性。 利用基因打靶的方法，我们获得了将原始的2F5重链可变区定向表达到小鼠重链可变区的小鼠(2F5 VH)，并进行了重要的观察，发现在前B期到未成熟的转变过程中，表达2F5重链的B细胞在骨髓中主要被删除。尽管存在这一严重缺陷，但外周仍存在少量但可分离的B细胞，它们保留了2F5 HC。 在这项应用中，我们建议全面扩展我们对2F5 VH小鼠的特征，首先验证这样的假设，即大多数剩余的外周B细胞已经失去了自身反应性，无论是通过LC编辑、无能还是对2F5 HC的修饰。使用一系列系统的遗传、免疫和药理学方法来调节自身耐受性，然后我们将检验这样的假设，即这些小鼠可以被推动产生高滴度的广泛中和抗体。最后，我们将通过使用一系列“反向突变”的2F5 VH小鼠(表达生殖系或具有不同选择的反向突变的2F5 VH区域)来扩展/完善我们在2F5 VH小鼠中的研究，以检验2F5前体B细胞谱系在免疫驱动的亲和力成熟后产生“2F5样”中和抗体的能力。 与公共卫生的相关性：基于抗体的保护性HIV-1疫苗的开发仍然是一个巨大的挑战。在这项研究中，我们建议使用我们设计的一系列小鼠株来表达广泛和有效的HIV-1抗体2F5的不同版本，以：1)确定自我耐受和亲和力成熟的过程如何影响B细胞产生中和抗体的能力，以及2)检查这些过程的适当操作是否可以诱导B细胞产生这种抗体。这些实验应该有助于制定新的策略，在患者中适当地诱导保护性HIV-1抗体。

DESCRIPTION (provided by applicant): The identification of a few rarely made antibodies to different HIV-1 envelope epitopes which are capable of neutralizing a broad range of HIV-1 isolates in vitro and controlling SHIV infection in vivo has provided hope for achieving an antibody-based HIV-1 vaccine strategy. One of these rare antibodies, 2F5, has particularly potent and broad neutralizing activity and is directed against an attractive antibody-based vaccine target, the MPER epitope of the HIV-1 gp41 region. 2F5 has unusually long charged CDR3s and exhibits polyreactivity, raising the possibility that antibodies like 2F5 cannot be elicited because the B cells from which they originate have reactivity with host antigens. Using a gene-targeting approach, we have generated mice (2F5 VH) with directed expression of the original 2F5 heavy chain (HC) variable region into the mouse HC locus, and made the important observation that 2F5 HC-expressing B cells are primarily deleted in the bone marrow at the pre-B to immature transition. Despite this profound defect, a small, but detactable population of B cells are present in the periphery which retain the 2F5 HC. In this application, we propose to comprehensively extend our characterization of 2F5 VH mice, first to test the hypothesis that most of their remaining peripheral B cells have lost autoreactivity, either by LC editing, anergy or modification of the 2F5 HC. Using a systematic array of genetic, immunization, and pharmacological approaches to modulate self-tolerance, we will then test the hypothesis that these mice can be pushed into producing high titers of broadly neutralizing antibodies. Finally, we will extend/refine our studies in 2F5 VH mice by using a series of "back-mutated" 2F5 VH mice (expressing 2F5 VH regions that are either germline or have different selected back-mutations), to examine the ability of the 2F5 precursor B cell repertoire, when shaped by immunization-driven affinity maturation, to generate "2F5-like" neutralizing antibodies. PUBLIC HEALTH RELEVANCE: The development of a protective antibody-based HIV-1 vaccine remains an enormous challenge. In this study, we propose to use a series of mouse strains we have engineered to express different versions of the broadly and potently neutralizing HIV-1 antibody 2F5 in order to: 1) determine how the processes of self-tolerance and affinity maturation impact the ability of B cells to make neutralizing antibodies, and 2) to examine if appropriate manipulation of these processes can elicit B cells to make such antibodies. These experiments should be helpful in formulating novel strategies for properly eliciting protective HIV-1 antibodies in patients.