Immunoglobulin CDR-H3 and neutralizing antibodies to HIV

性状（由申请方提供）：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突出。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很少在患者中产生[3;70]。这两种Nab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疏水H链互补决定区3（CDR-H3）。在以前的研究中，我们已经表明，具有疏水性CDR-H3的免疫球蛋白通常是从常规B细胞库中挑选的。因此，我们提出2F 5和4 E10样Nab难以引发，因为作为这种正常过程的结果，健康个体中这种类型的抗原结合位点的频率往往非常低。我们将检验使用长的疏水性CDR-H3间隔的强制富集将促进针对HIV-1 MPER的Nab的产生的假设。我们还建议测试互补假设，即在抗MPER Nab中使用这些长的疏水性CDR-H3间隔可能需要从正常耐受机制中释放。该申请响应RFA，因为其重点是HIV-1疫苗开发背景下B细胞和抗体调节的基础免疫学研究。我们提出的研究目标是HIV-1包膜糖蛋白gp 120/gp 41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起有效和强大的中和抗体应答。它们解决了在诱发Nabs中的耐受性和自身免疫性问题。而且，我们的研究促进了两名基础B细胞免疫学家施罗德和科尔尼博士以及两名艾滋病毒专家海恩斯和肖博士之间的合作。我们创造了一种老鼠，D-D ˇ FS，其中我们通过使B细胞使用疏水性DH阅读框，富含缬氨酸的DFL16.1 RF 2来强制使用疏水性CDR-H3。DFL16.1也比其他小鼠DH序列长2至4个密码子。因此，？D-D <$FS小鼠产生了多克隆库，其不仅富集了疏水性CDR-H3，而且还富集了更长的CDR-H3。为了测试长的疏水性CDR-H3在产生广泛中和的抗HIV抗体中的作用，施罗德博士将挑战突变体？D-D <$FS BALB/c小鼠，以及对照组？D-DFL和野生型BALB/c小鼠，使用从Haynes博士获得的MPER免疫原。为了开始测试耐受性的作用，Schroeder博士还将在BALB/c、C57 BL/6和C57 BL/6 sle 1/sle 2/sle 3同源背景下激发突变型和野生型DH基因座小鼠，每种小鼠均逐渐增加对自身反应性抗体产生的易感性。Dr.乔治Shaw将评价这些小鼠血清中是否存在中和抗体。Schroeder和Kearney博士将从表达中和MPER抗体的小鼠中产生杂交瘤，并获得单克隆抗体用于进一步研究。将进一步筛选这些抗MPER单克隆抗体的中和和自身反应性。将检查它们的结合特性。最后，将克隆和测序它们的V结构域以进一步关联中和与CDR-H3长度或疏水性之间的关系。事实证明，制造能够引发中和抗体的HIV疫苗比最初预期的要困难[7;44]。HIV包膜刺突在病毒感染中起关键作用，并且针对刺突基部的MPER区域的抗体具有广泛且有效的中和作用。不幸的是，这些抗体不仅非常罕见，而且往往是自身反应性的。这些抗体结合HIV的位点含有一种结合结构，该结合结构往往比其他抗体中的大多数这种结合结构更大且富含脂溶性氨基酸。我们已经使用遗传技术来迫使小鼠表达富含更长结合结构以及更长、更脂溶性结合结构的抗体。为了测试这些特征在多大程度上能够产生中和MPER抗体，我们建议用HIV MPER免疫原攻击我们的突变小鼠，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相关联。

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.