Immunoglobulin CDR-H3 and neutralizing antibodies to HIV

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（nab）中，2F5和4E10因其效力和广泛中和活性而脱颖而出。这两种抗体都针对gp41茎的膜近端外区（MPER）。不幸的是，2F5和4e10样抗mper抗体在患者中很少被激发[3；70]。这两种nab与常规nab的不同之处在于，它们的每个抗原结合位点都含有一个长长的疏水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-H3s的免疫球蛋白通常从常规B细胞库中剔除。因此，我们认为2F5-和4e10样nab很难被诱导，因为这种正常过程的结果是，这种类型的抗原结合位点在健康个体中的频率往往很低。我们将检验这一假设，即使用长疏水性CDR-H3间隔进行强制富集将促进针对HIV-1 MPER的nab的产生。我们还建议验证一个补充假设，即在抗mper抗体中使用这些长疏水性CDR-H3间隔可能需要从正常耐受机制中释放出来。该申请响应RFA，因为其重点是在HIV-1疫苗开发背景下对B细胞和抗体调节的基础免疫学研究。我们提出的研究针对HIV-1包膜糖蛋白gp120/gp41的一个关键保守表位。他们的重点是阐明基本的免疫机制，通过这种机制，可以在未感染的个体中引发有效和强大的中和抗体反应。它们解决了在诱导抗体时的耐受性和自身免疫问题。而且，我们的研究促进了两位基础B细胞免疫学家之间的合作。施罗德和科尔尼；和两位艾滋病专家，dr。海恩斯和肖事务所。我们创造了一种老鼠，？D-D¿FS，其中我们通过将B细胞引入疏水DH阅读框（富含缬氨酸的DFL16.1 RF2）来强制使用疏水CDR-H3s。DFL16.1也比其他小鼠DH序列长2 ~ 4个密码子。因此，？D-D¿FS小鼠创建了一个多克隆库，不仅丰富了疏水性CDR-H3s，而且也丰富了更长的CDR-H3s。为了测试长，疏水性CDR-H3s在允许产生广泛中和的抗hiv抗体方面的作用，Schroeder博士将挑战突变体？D-D /c小鼠，以及对照组？D-DFL和野生型BALB/c小鼠，使用Haynes博士获得的MPER免疫原。为了开始测试耐受性的作用，Schroeder博士还将挑战具有BALB/c、C57BL/6和C57BL/6 sle1/sle2/sle3基因背景的突变型和野生型DH位点小鼠，每一种基因背景都会逐渐增加对自身反应性抗体产生的易感性。乔治·肖医生会评估这些老鼠的血清中是否存在中和抗体。Drs。Schroeder和Kearney将从这些表达中和性MPER抗体的小鼠中产生杂交瘤，并获得单克隆抗体供进一步研究。这些抗mper单克隆抗体将进一步筛选中和和自身反应性。我们将研究它们的绑定特性。最后，他们的V结构域将被克隆和测序，以进一步关联中和和CDR-H3长度或疏水性之间的关系。事实证明，研制能够产生艾滋病毒中和抗体的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着关键作用，针对刺突底部MPER区域的抗体被广泛而有效地中和。不幸的是，这些抗体不仅极为罕见；它们也倾向于自我反应。这些抗体结合HIV的位点包含一个结合结构，与其他抗体中的大多数这种结合结构相比，这种结合结构往往更大，而且富含脂溶性氨基酸。我们已经使用遗传技术迫使小鼠表达针对较长结合结构和更长、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性MPER抗体，我们建议用HIV MPER免疫原刺激突变小鼠，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.