Immunoglobulin CDR-H3 and neutralizing antibodies to HIV

描述(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因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样的抗MPER NAB在患者中很少发生[3；70]。这两种NAb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疏水H链互补决定区3(CDR-H3)。在以前的研究中，我们已经证明，具有疏水性CDR-H3s的免疫球蛋白通常是从传统的B细胞谱系中剔除的。因此，我们认为2F5和4E10样的NAB是很难诱导的，因为作为这一正常过程的结果，这种类型的抗原结合位点在健康个体中的频率往往非常低。我们将检验这样的假设，即强制浓缩使用长的疏水CDR-H3间隔将促进针对HIV-1 MPER的NAB的产生。我们还建议检验互补假设，即在抗MPER NAB中使用这些长的、疏水的CDR-H3间隔可能需要从正常的耐受机制中释放。该应用是对RFA的响应，因为它的重点是B细胞的基础免疫学研究和HIV-1疫苗开发背景下的抗体调节。我们建议的研究针对HIV-1包膜糖蛋白gp120/gp41上的一个关键保守表位。他们专注于阐明基本的免疫机制，通过这些机制，可以在未感染的个体中引发有效和强大的中和抗体反应。它们解决了引起NAB的耐受性和自身免疫的问题。而且，我们的研究促进了两位著名的基础B细胞免疫学家施罗德博士和科尔尼博士以及两位艾滋病毒专家海恩斯博士和肖博士之间的合作。我们已经创造了一种小鼠，？D-D？FS，在那里，我们通过让B细胞使用疏水的DH读框，富含Valine的DFL16.1RF2，强制使用疏水性CDR-H3s。DFL16.1也比其他小鼠的DH序列长2到4个密码子。因此，D-D？FS小鼠创造了一种多克隆谱系，不仅丰富了疏水性CDR-H3s，而且还丰富了更长的CDR-H3s。为了测试长的、疏水的CDR-H3在允许产生广泛中和抗HIV抗体方面的作用，Schroeder博士将用从Haynes博士获得的MPER免疫原挑战突变的D-D？FS BALB/c小鼠以及对照的D-DFL和野生型BALB/c小鼠。为了开始测试耐受性的作用，施罗德博士还将在BALB/c、C57BL/6和C57BL/6sle1/sle2/sle3同源基因背景上挑战突变和野生型DH基因座小鼠，每种背景都会逐渐增加产生自身反应性抗体的易感性。乔治·肖博士将评估这些小鼠的血清中是否存在中和抗体。施罗德和科尔尼博士将从那些表达中和MPER抗体的小鼠身上产生杂交瘤，并获得用于进一步研究的克隆抗体。这些抗MPER的单抗将进一步筛选中和和自身反应性。我们将检查它们的绑定属性。最后，我们将对它们的V结构域进行克隆和测序，以进一步研究中和作用与CDR-H3长度或疏水性的关系。事实证明，创造能够诱导HIV中和抗体的疫苗比最初预期的要困难[7；44]。HIV包膜尖峰在病毒感染中起着关键作用，针对尖峰底部MPER区域的抗体具有广泛而有效的中和作用。不幸的是，这些抗体不仅极其罕见，而且往往具有自身反应性。这些抗体与HIV结合的部位包含一种结合结构，与其他抗体中的大多数此类结合结构相比，这种结合结构往往更大，而且富含脂溶氨基酸。我们已经使用遗传技术迫使小鼠表达富含更长结合结构以及更长更脂溶结合结构的抗体。为了测试这些功能将在多大程度上能够产生中和MPER抗体，我们建议用HIV MPER免疫原挑战我们的突变小鼠，分离抗MPER抗体，测试这些抗体是否中和HIV，并进一步将结构与功能联系起来。

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 elicited in patients [3;70]. Both of these Nabs differ from the norm in that each of their antigen binding sites contain a long, hydrophobic H chain complementarity determining region 3 (CDR-H3). In previous studies we have shown that immunoglobulins with hydrophobic CDR-H3s are normally culled from the conventional B cell repertoire. We thus propose that 2F5- and 4E10-like Nabs are difficult to elicit because as a result of this normal process the frequency of this type of antigen binding site in healthy individuals tends to be very low. We will test the hypothesis that forced enrichment for use of long, hydrophobic CDR-H3 intervals will promote the production of Nabs directed against the HIV-1 MPER. We also propose to test the complementary hypothesis that the use of these long, hydrophobic CDR-H3 intervals in anti-MPER Nabs may require release from normal tolerance mechanisms. The application is responsive to the RFA in that its focus is basic immunology research on B cell and antibody regulation in the context of HIV-1 vaccine development. Our proposed studies target a critical conserved epitope on the HIV-1 envelope glycoprotein gp120/gp41. They focus on elucidating basic immune mechanisms by which an effective and robust neutralizing antibody response can be elicited in uninfected individuals. They address the issue of tolerance and autoimmunity in eliciting Nabs. And, our studies foster collaboration between two established basic B cell immunologists, Drs. Schroeder and Kearney; and two experts in HIV, Drs. Haynes and Shaw. We have created a strain of mouse, ?D-D¿FS, where we force use of hydrophobic CDR-H3s by committing B cells to the use of a hydrophobic DH reading frame, valine-enriched DFL16.1 RF2. DFL16.1 is also two to four codons longer than the other mouse DH sequences. Thus, the ?D-D¿FS mouse creates a polyclonal repertoire that is not only enriched for hydrophobic CDR-H3s, but for longer CDR-H3s as well. To test the role of long, hydrophobic CDR-H3s in permitting the generation of broadly neutralizing anti-HIV antibodies, Dr. Schroeder will challenge mutant ?D-D¿FS BALB/c mice, as well as control ?D-DFL and wild-type BALB/c mice, with MPER immunogens obtained from Dr. Haynes. To begin to test the role of tolerance, Dr. Schroeder will also challenge mutant and wild-type DH locus mice on BALB/c, C57BL/6, and C57BL/6 sle1/sle2/sle3 congenic backgrounds, each of which progressively increases susceptibility to the generation of autoreactive antibodies. Dr. George Shaw will evaluate the sera from these mice for the presence of neutralizing antibodies. Drs. Schroeder and Kearney will generate hybridomas from those mice that express neutralizing MPER antibodies and obtain monoclonal antibodies for further study. These anti-MPER monoclonal antibodies will be further screened for neutralization and for autoreactivity. Their binding properties will be examined. Finally, their V domains will be cloned and sequenced to further correlate the relationship between neutralization and CDR-H3 length or hydrophobicity. The creation of vaccines capable of eliciting neutralizing antibodies to HIV has proven more difficult than first expected [7;44]. The HIV envelope spike plays a key role in viral infection, and antibodies directed against the MPER region at the base of the spike are broadly and potently neutralizing. Unfortunately, these antibodies are not only extremely uncommon; they also tend to be autoreactive. The site at which these antibodies bind HIV contains a binding structure that tends to be both larger and enriched for fat soluble amino acids than most such binding structures in other antibodies. We have used genetic techniques to force mice to express antibodies enriched for longer binding structures as well as for longer, more fat soluble binding structures. To test the extent to which these features will enable generation of neutralizing MPER antibodies, we propose to challenge our mutant mice with HIV MPER immunogens, isolate anti-MPER antibodies, test whether these antibodies neutralize HIV, and further correlate structure with function.